Carbazole compounds and therapeutic uses of the compounds

ABSTRACT

Compounds of the general structural formula (I) and (II) and use of the compounds and salts and hydrates thereof, as therapeutic agents are disclosed. Treatable diseases and conditions include cancers, inflammatory diseases and conditions, and immunodeficiency diseases. (I), (II).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/231,841, filed Apr. 1, 2014, which is a continuation of U.S.application Ser. No. 13/121,051, which is a U.S. National PhaseApplication of PCT/US2009/059558, filed Oct. 5, 2009, which claims thebenefit of and priority to U.S. Provisional Application No. 61/102,913,filed Oct. 6, 2008, each of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

This invention relates to carbazole compounds, to methods of preparingthe compounds, to pharmaceutical compositions containing the compounds,and to their use as therapeutic agents. In particular, the inventionrelates to carbazole compounds and their use in a variety of therapeuticareas, including the treatment of cancers.

BACKGROUND OF THE INVENTION

The frequency of cancer in humans has increased in the developed worldas the population has aged. For some types of cancers and the stage ofdisease at diagnosis, morbidity and mortality rates have not improvedsignificantly in recent years despite extensive research. Induction ofcell death is one of the most attractive cancer treatment strategies.There is a significant need to identify agents that are capable ofinducing cell death in tumor cells and/or that potentiatechemotherapeutic and radiation therapies.

SUMMARY OF THE INVENTION

The present invention is directed to compounds and compositions thatinduce cell death, and to therapeutic uses of the compounds in thetreatment of a cancer and other conditions in individuals in need ofsuch treatment. The present invention also is directed to methods ofpreparing the therapeutic compounds.

More particularly, the present invention is directed to compounds andmethods of treating diseases and conditions such as cancers,inflammatory diseases, microbial infections, viral infections, andprotozoan infections. The compounds are useful in a method comprisingadministering a therapeutically effective amount of a compound ofstructural formula (I) to an individual in need thereof.

In particular, the present invention is directed to carbazole compoundshaving a structural formula (I):

wherein R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl, C₁₋₆ haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,OR^(e), N(R^(e))₂, and SR^(e); alternatively, either R^(a) and R¹ orNR^(e) and R¹ together with the carbon atoms to which they are attachedform a five or six-membered aliphatic carbocyclic or heterocyclic ring;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,C₁₋₆ haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e),N(R^(e))₂, and SR^(e), alternatively, either R^(b) and R⁶ or NR^(e) andR⁶ together with the carbon atoms to which they are attached form a fiveor six-membered aliphatic carbocyclic ring or a five or six-memberedaliphatic carbocyclic or heterocyclic ring;

R^(c) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,C₁₋₆hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, andC(═O)R^(e), or R^(c) and R^(d) are taken together to form a five, six,or seven-membered aliphatic ring, optionally containing an oxygen atom;

R^(d) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(e), or R^(d)and R⁷ together with the atoms to which they are attached form a five orsix-membered aliphatic ring;

R^(e), independently, is selected from the group consisting of hydrogen,C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or twoR^(e) groups taken together with a nitrogen to which they are attachedto form a five or six-membered aliphatic ring;

R¹, R², R³, R⁴, R⁵, and R⁶, independently, are selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, halo, OR^(e), C(═O)R^(e), C(═O)OR^(e), OC(═O)R^(e),C(═O)N(R^(e))₂, C(═O)NR^(e)SO₂R^(e), N(R^(e))₂, NR^(e)C(═O)R^(e),NR^(e)C(═O)N(R^(e))₂, CN, NO₂, CF₃, OCF₃, SR^(e), SOR^(e), SO₂R^(e),SO₂N(R^(e))₂, and OSO₂CF₃;

R⁷ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and

n is 0, 1, 2, 3, 4, or 5,

or a pharmaceutically acceptable salt or hydrate thereof.

The present invention also is directed to carbazole compounds having astructural formula (II):

wherein R^(f) is selected from the group consisting of hydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(h),or R^(f) and R^(g) are taken together to form a five, six, orseven-membered aliphatic ring optionally containing an oxygen atom;

R^(g) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(h), or R^(g)and R⁸ together with the atoms to which they are attached form a five orsix-membered aliphatic ring;

R^(h), independently, is selected from the group consisting of hydrogen,C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or twoR^(h) groups taken together with a nitrogen to which they are attachedto form a five or six-membered aliphatic ring;

R⁸ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;

R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴, independently, are selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, halo, OR^(h), C(═O)R^(h), C(═O)OR^(h), OC(═O)R^(h),C(═O)N(R^(h))₂, C(═O)NR^(h)SO₂R^(h), N(R^(h))₂, NR^(e)C(═O)R^(h),NR^(h)C(═O)N(R^(h))₂, CN, NO₂, CF₃, OCF₃, SR^(h), SOR^(h), SO₂R^(h),SO₂N(R^(h))₂, and OSO₂CF₃;

p is 0, 1, 2, 3, 4, or 5,

with the proviso that when p is 2, one of R^(f) and R^(g) is differentfrom ethyl, or a pharmaceutically acceptable salt or hydrate thereof.

A disease or condition that can be treated in accordance with presentinvention includes, for example, a cancer, inflammation, autoimmunedisease, microbial infection, protozoan infection, viral infection,graft versus host disease, a condition associated with HIV infection, orpre-cancerous cells. Forms of cancer that can be treated include, butare not limited to, renal cell carcinoma, sarcoma, prostate cancer,breast cancer, pancreatic cancer, myeloma, myeloid and lymphoblasticleukemia, neuroblastoma, glioblastoma, or a cancer caused by HTLVinfection.

In some embodiments, the compound has a general structural formula (Ia):

wherein R^(a) is C₁₋₃ alkyl, C₁₋₄ haloalkyl, C₃₋₅ cycloalkyl, N(R^(e))₂,or OR^(e), or R^(a) and R¹ together with the carbon atoms to which theyare attached form a five or six-membered aliphatic carbocyclic ring;

R^(b) is C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₃₋₅ cycloalkyl, N(R^(e))₂, orOR^(e), or R^(b) and R⁶ together with the carbon atoms to which they areattached form a five or six-membered aliphatic carbocyclic ring or afive or six-membered aliphatic ring containing one nitrogen atom;

R^(c) is C₁₋₆ alkyl, C₃₋₅cycloalky, or C₁₋₃hydroxyalkyl;

R^(d) is hydrogen, C₁₋₄ alkyl, or C₃₋₅ cycloalkyl, or R^(d) and R⁷together with the atoms to which they are attached form a five orsix-membered aliphatic ring containing one nitrogen atom, or R^(c) andR^(d) are taken together to form a six- or seven-membered aliphaticring, optionally containing an oxygen atom;

R^(e), independently, is hydrogen or C₁₋₃ alkyl;

R¹ is hydrogen or C₁₋₃ alkyl;

R² is hydrogen, hydroxy, or C₁₋₃ alkoxy;

R³ and R⁴, independently, are hydrogen or C₁₋₃ alkyl;

R⁵ is hydrogen, hydroxy, C₁₋₃alkoxy, or halo;

R⁶ is hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, or halo;

R⁷ is hydrogen or C₁₋₃ alkyl; and

n is 0, 1, 2, 3, 4, or 5,

or a pharmaceutically acceptable salt or hydrate thereof.

In other embodiments, the compound has a general structural formula(Ib):

wherein R^(a) is methyl, ethyl, n-propyl, cyclopropyl, NH(CH₃), or OCH₃,or R^(a) and R¹ together with the carbon atoms to which they areattached form a five-membered aliphatic carbocyclic ring;

R^(b) is methyl, ethyl, n-propyl, cyclopropyl, NH(CH₃), or OCH₃, orR^(b) and R⁶ together with the carbon atoms to which they are attachedform a five-membered aliphatic carbocyclic ring or a five-memberedaliphatic ring containing one nitrogen atom;

R^(c) is methyl, ethyl, n-propyl, isopropyl, cyclobutyl, or2-hydroxyethyl;

R^(d) is hydrogen, methyl, ethyl, or cyclobutyl, or R^(d) and R⁷together with the atoms to which they are attached form a five-memberedaliphatic ring containing one nitrogen atom; or R^(c) and R^(d) aretaken together to form a morpholino moiety; a tetrahydrofuryl moiety; apiperidinyl moiety; a

moiety, or a

moiety;

R¹ is hydrogen;

R² is hydrogen, hydroxy, or methoxy;

R³ and R⁴ are hydrogen;

R⁵ is hydrogen, hydroxy, methoxy, or fluoro;

R⁶ is hydrogen, methyl, methoxy or fluoro;

R⁷ is hydrogen; and

n is 1 or 2,

or a pharmaceutically acceptable salt or hydrate thereof.

In other embodiments, the compound has a general structural formula(IIa):

wherein R^(f) is C₁₋₆ alkyl;

R^(g) is hydrogen or C₁₋₄ alkyl, or R^(g) and R⁸ together with the atomsto which they are attached form a five or six-membered aliphatic ringcontaining one nitrogen atom;

R⁹ is hydrogen or C₁₋₃ alkyl;

R¹⁰ is hydrogen, hydroxy, or C₁₋₃ alkoxy;

R¹¹ and R¹², independently, are hydrogen or C₁₋₃ alkyl;

R¹³ is hydrogen, hydroxy, C₁₋₃alkoxy, or halo;

R¹⁴ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkoxy;

R⁸ is hydrogen or C₁₋₃ alkyl; and

p is 0, 1, 2, 3, 4, or 5,

with the proviso that when p is 2, one of R^(f) and R^(g) is differentfrom ethyl,

or a pharmaceutically acceptable salt or hydrate thereof.

In still other embodiments, the compound has a structural formula (IIb):

wherein R^(f) is methyl or ethyl;

R^(g) is hydrogen or methyl or R^(g) and R⁸ together with the atoms towhich they are attached form a five-membered aliphatic ring containingone nitrogen atom;

R⁸ is hydrogen; and

p is 1 or 2,

or a pharmaceutically acceptable salt or hydrate thereof.

One aspect of the present invention is to provide a method of treating acondition or disease by administering a therapeutically effective amountof one or more compound of structural formula (I), (Ia), (Ib), (II),(IIa), or (IIb), or a composition comprising one or more of a compoundof structural formula (I), (Ia), (Ib), (II), (IIa), or (IIb), to anindividual in need thereof. The composition can further comprise a deathreceptor activator of a TNF family polypeptide. The activator can be aTNF polypeptide, such as one or more of NGF, CD40L, CD137L/4-1BBL,TNF-α, CD134L/OX40L, CD27L/CD70, FasL/CD95, CD30L, TNF-β/LT-α, LT-β, andTRAIL.

Another aspect of the present invention is to provide pharmaceuticalcompositions comprising one or more compound of structural formula (I)or (II), and use of the compositions in a therapeutic treatment of adisease or condition.

Yet another aspect of the present invention is to provide a method oftreating an individual undergoing a chemotherapeutic or radiotherapeutictreatment for a medical condition comprising administration of acompound of structural formula (I) and/or (II) in combination with achemotherapeutic agent, a radiotherapeutic agent, or both, to theindividual. A nonlimiting indication treated by this method is a cancer.

The above and additional aspects of the present invention will becomeapparent from the following nonlimiting detailed description ofpreferred embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a plot of fold to NF-κB activity relative to DMSO control vs.concentration for carbazoles of the present invention;

FIG. 1B is a plot of EC₅₀ (μM) for p53 activation and NF-κB inhibitionfor carbazoles of the present invention;

FIGS. 2A through 2K are plots of % cell viability vs. concentration (μM)for various tumor cells treated with carbazoles of the presentinvention;

FIG. 3 contains a plot of tumor volume vs. days of treatment in an HCT116 sc xenograft model using the compound of Example 7;

FIG. 4 is a schematic showing the three dimensional analysis of activecarbazole compounds;

FIG. 5 is a schematic showing the three dimensional analysis of inactivecarbazole compounds;

FIG. 6 is a schematic showing the three dimensional structure of anactive carbazole compound, i.e., Example 2;

FIG. 7 is a schematic showing the three dimensional structure of aninactive carbazole compound, i.e., Compound 200;

FIGS. 8A-B contain plots of tumor volume (mm³) vs. days of treatment forindividual tumor growth in mice treated with a control vehicle (FIG. 8A)and in mice treated with the compound of Example 7 (FIG. 8B);

FIGS. 9A-C contain plots of tumor volume (mm³) vs. days of treatmentwith a control vehicle (FIG. 9A) and with the compound of Example 7(FIG. 9B and FIG. 9C);

FIGS. 10A-B contain plots of the relative weight of individual mice vs.days after cell inoculation in mice treated with the control vehicle(FIG. 10A) and mice treated with the compound of Example 7 (FIG. 10B);and

FIG. 11 contains plots of concentration of Compound 100 (μM) vs.relative cell survival for thirteen cancer cell lines showing that apresent carbazole compound is an effective agent against numerous typesof cancer;

FIG. 12 contains bar graphs showing the antiparasitic activity ofvarious carbazole compounds against Plasmodium falciparum(strain D10);and

FIGS. 13A-B contain plots showing the antibacterial activity of variouscarbazole compounds against Gram negative (FIG. 13A) and Gram positivebacteria (FIG. 13B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

With respect to the compounds, compositions, and methods disclosedherein, the terminology used is for the purpose of describing particularembodiments and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

The present invention is directed to compounds having a generalstructural formula (I) and (II). The carbazole compounds disclosedherein are useful in the treatment of diseases and conditions, such ascancers, inflammatory disease, microbial infections, viral infections,protozoan infections, or an autoimmune disease.

wherein R^(a) is selected from the group consisting of hydrogen,C₁₋₆alkyl, C₁₋₆haloalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR^(e), N(R^(e))₂, and SR^(e), or either R^(a) and R¹ orNR^(e) and R¹ together with the carbon atoms to which they are attachedform a five or six-membered aliphatic carbocyclic or heterocyclic ring;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₁₋₆haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e),N(R^(e))₂, and SR^(e), or either R^(b) and R⁶ or NR^(e) and R⁶ togetherwith the carbon atoms to which they are attached form a five orsix-membered aliphatic carbocyclic ring or a five or six-memberedaliphatic carbocyclic or heterocyclic ring;

R^(c) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, andC(═O)R^(e), or R^(c) and R^(d) are taken together to form a five, six,or seven-membered aliphatic ring, optionally containing an oxygen atom;

R^(d) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(e), or R^(d)and R⁷ together with the atoms to which they are attached form a five orsix-membered aliphatic ring;

R^(e), independently, is selected from the group consisting of hydrogen,C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or twoR^(e) groups taken together with a nitrogen to which they are attachedto form a five or six-membered aliphatic ring;

R¹, R², R³, R⁴, R⁵, and R⁶, independently, are selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, halo, OR^(e), C(═O)R^(e), C(═O)OR^(e), OC(═O)R^(e),C(═O)N(R^(e))₂, C(═O)NR^(e)SO₂R^(e), N(R^(e))₂, NR^(e)C(═O)R^(e),NR^(e)C(═O)N(R^(e))₂, CN, NO₂, CF₃, OCF₃, SR^(e), SOR^(e), SO₂R^(e),SO₂N(R^(e))₂, and OSO₂CF₃;

R⁷ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and

n is 0, 1, 2, 3, 4, or 5,

or a pharmaceutically acceptable salt or hydrate thereof.

In preferred embodiments, the compounds have general structural formula(Ia):

wherein R^(a) is C₁₋₃ alkyl, C₁₋₄ haloalkyl, C₃₋₅ cycloalkyl, N(R^(e))₂,or OR^(e), or R^(a) and R¹ together with the carbon atoms to which theyare attached form a five or six-membered aliphatic carbocyclic ring;

R^(b) is C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₃₋₅ cycloalkyl, N(R^(e))₂, orOR^(e), or R^(b) and R⁶ together with the carbon atoms to which they areattached form a five or six-membered aliphatic carbocyclic ring or afive or six-membered aliphatic ring containing one nitrogen atom;

R^(c) is C₁₋₆ alkyl, C₃₋₅ cycloalkyl, or C₁₋₃ hydroxyalkyl;

R^(d) is hydrogen, C₁₋₄ alkyl, or C₃₋₅cycloalkyl, or R^(d) and R⁷together with the atoms to which they are attached form a five orsix-membered aliphatic ring containing one nitrogen atom or R^(c) andR^(d) are taken together to form a six or seven-membered aliphatic ring,optionally containing an oxygen atom;

R^(e), independently, is hydrogen or C_(1-s) alkyl;

R¹ is hydrogen or C₁₋₃ alkyl;

R² is hydrogen, hydroxy, or C₁₋₃ alkoxy;

R³ and R⁴, independently, are hydrogen or C₁₋₃ alkyl;

R⁵ is hydrogen, hydroxy, C₁₋₃alkoxy, or halo;

R⁶ is hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, or halo;

R⁷ is hydrogen or C₁₋₃ alkyl; and

n is 0, 1, 2, 3, 4, or 5,

or a pharmaceutically acceptable salt or hydrate thereof.

In more preferred embodiments, the compounds have a general structuralformula (Ib):

wherein R^(a) is methyl, ethyl, n-propyl, cyclopropyl, NH(CH₃), or OCH₃,or R^(a) and R¹ together with the carbon atoms to which they areattached form a five-membered aliphatic carbocyclic ring;

R^(b) is methyl, ethyl, n-propyl, cyclopropyl, NH(CH₃), or OCH₃, orR^(b) and R⁶ together with the carbon atoms to which they are attachedform a five-membered aliphatic carbocyclic ring or a five-memberedaliphatic ring containing one nitrogen atom;

R^(e) is methyl, ethyl, n-propyl, isopropyl, cyclobutyl, or2-hydroxyethyl;

R^(d) is hydrogen, methyl, ethyl, or cyclobutyl, or R^(d) and R⁷together with the atoms to which they are attached form a five-memberedaliphatic ring containing one nitrogen atom, or R^(c) and R^(d) aretaken together to form a morpholino moiety, a tetrahydrofuryl moiety, apiperidinyl moiety, or a

moiety, a

moiety;

R¹ is hydrogen;

R² is hydrogen, hydroxy, or methoxy;

R³ and R⁴ are hydrogen;

R⁵ is hydrogen, hydroxy, methoxy, or fluoro;

R⁶ is hydrogen, methyl, methoxy, or fluoro;

R⁷ is hydrogen; and

n is 1 or 2,

or a pharmaceutically acceptable salt or hydrate thereof.

In another embodiment, the present invention also is directed tocarbazole compounds having a structural formula (II):

wherein R^(f) is selected from the group consisting of hydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(h),or R^(f) and R^(g) are taken together to form a five, six, orseven-membered aliphatic ring optionally containing an oxygen atom;

R^(g) is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(h), or R^(g)and R^(h) together with the atoms to which they are attached form afive, six, or seven-membered aliphatic ring;

R^(h), independently, is selected from the group consisting of hydrogen,C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or twoR^(h) groups taken together with a nitrogen to which they are attachedto form a five or six-membered aliphatic ring;

R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴, independently, are selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, halo, OR^(h), C(═O)R^(h), C(═O)OR^(h), OC(═O)R^(h),C(═O)N(R^(h))₂, C(═O)NR^(h)SO₂R^(h), N(R^(h))₂, NR^(h)C(═O)R^(h),NR^(h)C(═O)N(R^(h))₂, CN, NO₂, CF₃, OCF₃, SR^(h), SOR^(h), SO₂R^(h),SO₂N(R^(h))₂, and OSO₂CF₃;

R⁸ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and

p is 0, 1, 2, 3, 4, or 5,

with the proviso that when p is 2, one of R^(f) and R^(g) is differentfrom ethyl,

or a pharmaceutically acceptable salt or hydrate thereof.

In other embodiments, the compound has a general structural formula(IIa):

wherein R^(f) is C₁₋₆ alkyl;

R⁹ is hydrogen or C₁₋₄ alkyl, or R^(g) and R⁸ together with the atoms towhich they are attached form a five or six-membered aliphatic ringcontaining one nitrogen atom;

R⁹ is hydrogen or C₁₋₃ alkyl;

R¹⁰ is hydrogen, hydroxy, or C₁₋₃ alkoxy;

R¹¹ and R¹², independently, are hydrogen or C₁₋₃ alkyl;

R¹³ is hydrogen, hydroxy, C₁₋₃alkoxy, or halo;

R¹⁴ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkoxy;

R⁸ is hydrogen or C₁₋₃ alkyl; and

p is 0, 1, 2, 3, 4, or 5,

with the proviso that when p is 2, one of R^(f) and R^(g) is differentfrom ethyl,

or a pharmaceutically acceptable salt or hydrate thereof.

In still other embodiments, the compound has a structural formula (IIb):

wherein R^(f) is methyl or ethyl;

R^(g) is hydrogen or methyl, or R^(g) and R⁸ together with the atoms towhich they are attached form a five-membered aliphatic ring containingone nitrogen atom;

R⁸ is hydrogen; and

p is 1 or 2,

or a pharmaceutically acceptable salt or hydrate thereof.

As used herein, the term “alkyl” means straight chained and branchedhydrocarbon groups containing the indicated number of carbon atoms,typically methyl, ethyl, and straight chain and branched propyl andbutyl groups. The term “cycloalkyl” is defined as a cyclic hydrocarbongroup containing the indicated number of carbon atoms, e.g.,cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

The term “heterocycloalkyl” means monocyclic, bicyclic, and, tricycliccycloalkyl groups containing one or more heteroatoms selected from thegroup consisting of oxygen, nitrogen, and sulfur in the ring structure.A “heterocycloalkyl” group also can contain an oxo group (═O) attachedto the ring. Nonlimiting examples of heterocycloalkyl groups include,but are not limited to, 1,3-dioxolane, 2-pyrazoline, pyrazolidine,pyrrolidine, piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine,thiopholine, piperidine, 1,4-dithiane, and 1,4-dioxane.

The term “halo” or “halogen” means fluorine, bromine, chlorine, andiodine.

The term “haloalkyl” means an alkyl group substituted with one or more,e.g., 1 to 3, halo substituents, either fluoro, chloro, bromo, iodo, orcombinations thereof. Similarly, “halocycloalkyl” is defined as acycloalkyl group having one or more halo substituents.

The term “aryl,” alone or in combination, means a monocyclic orpolycyclic aromatic group, preferably a monocyclic or bicyclic aromaticgroup, e.g., phenyl or naphthyl. Unless otherwise indicated, an “aryl”group can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, halo, alkyl, hydroxyalkyl, alkoxy,alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio,alkylsulfinyl, and alkylsulfonyl. Exemplary aryl groups include phenyl,naphthyl, tetrahydronaphthyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl,3-trifluoromethylphenyl, 4-nitrophenyl, and the like.

The term “heteroaryl” means a monocyclic or bicyclic ring systemcontaining one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring, and which can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents, like halo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl,isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl,pyrimidinyl, thiazonyl, and thiadiazolyl.

The term “alkylene” means an alkyl group having a substituent. Forexample, the term “C₁₋₃alkylenearyl” refers to an alkyl group containingone to three carbon atoms and substituted with an aryl group.

The term “hydroxy” means —OH.

The term “alkoxy” means —OR, wherein R is alkyl.

The term “amino” means —NH₂, and the term “alkylamino” means —NR₂,wherein at least one R is alkyl and the second R is alkyl or hydrogen.

The term “acylamino” means R(═O)N—, wherein R is alkyl or aryl.

The term “alkylthio” means —SR, wherein R is alkyl.

The term “nitro” means —NO₂.

The term “trifluoromethyl” means —CF₃.

The term “trifluoromethoxy” means —OCF₃.

The term “cyano” means —CN.

The term “alkoxyalkyl” means an alkyl group wherein a hydrogen has beenreplaced by an alkoxy group.

The term “hydroxyalkyl” means an alkyl group wherein a hydrogen hasreplaced by a hydroxy group.

The term “alkylsulfinyl” means R—SO₂—, wherein R is alkyl.

The term “alkylsulfonyl” means R—SO₃—, wherein R is alkyl.

The term “morpholino moiety” means

The term “tetrahydrofuryl moiety” means

The term “piperidinyl moiety” means

optionally substituted with an —OH or —CH₂OH group.

The terms “effective amount” and “therapeutically effective amount,”when used in reference to a compound or composition, means a sufficientamount of the compound or composition to provide the desired result. Theexact amount desired or required will vary depending on the particularcompound or composition used, its mode of administration, and the like.Thus, it is not always possible to specify an exact “effective amount”or “therapeutically effective amount”. However, an appropriate effectiveamount can be determined by one of ordinary skill in the art informed bythe instant disclosure using only routine experimentation.

The term “suitable” means an entity, e.g., a moiety, substituent, orcompound, that is compatible with the compounds or compositions asprovided herein for the stated purpose. Suitability for the statedpurpose may be determined by one of ordinary skill in the art using onlyroutine experimentation.

The term “administer”, when used to describe the dosage of a compound orcomposition, means a single dose or multiple doses of the compound orcomposition.

“In vivo” means within a living subject, as within an animal or human.In this context, agents can be used therapeutically in a subject totreat a condition or disease, or a symptom thereof. The agents also canbe used as a prophylactic to prevent the occurrence or recurrence of adisease conditions or symptoms associated therewith.

“Ex vivo” means outside a living subject. Examples of ex vivo cellpopulations include in vitro cell cultures and biological samples suchas fluid or tissue samples from humans or animals. Such samples can beobtained by methods well known in the art. Exemplary biological fluidsamples include blood, cerebrospinal fluid, urine, saliva. Exemplarytissue samples include tumors and biopsies thereof. In this context, thepresent compounds can be in numerous applications, both therapeutic andexperimental.

The term “radiosensitizer” means a compound administered to a human orother animal in a therapeutically effective amount to increase thesensitivity of cells to electromagnetic radiation and/or to promote thetreatment of diseases treatable with electromagnetic radiation.

The terms “electromagnetic radiation” and “radiation” mean, but are notlimited to, radiation having the wavelength of 10⁻²⁰ to 100 meters.

The term “cell death” means a process wherein cell functioning,proliferation, and metabolism is stopped.

The term “cancer treatment” means any treatment for cancer known in theart including, but not limited to, chemotherapy and radiation therapy.

The term “combination with”, when used to described administration of apresent carbazole compound and any additional treatment, means that thecarbazole compound can be administered prior to, simultaneously with, orafter the additional treatment, or a combination thereof.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to eliminating, reducing, or ameliorating a disease or conditionand/or symptoms associated therewith. Although not precluded, treating adisease or condition does not require that the disease, condition, orsymptoms associated therewith be completely eliminated. As used herein,the terms “treat,” “treating,” “treatment,” and the like may include“prophylactic treatment,” which refers to reducing the probability ofredeveloping a disease or condition, or of a recurrence of apreviously-controlled disease or condition, in a subject who does nothave, but is at risk of or is susceptible to, redeveloping a disease orcondition or a recurrence of the disease or condition. The term “treat”and synonyms contemplate administering a compound of the invention to anindividual in need of such treatment.

Within the meaning of the invention, “treatment” also includes relapseprophylaxis or phase prophylaxis, as well as the treatment of acute orchronic signs, symptoms and/or malfunctions. The treatment can beorientated symptomatically, for example, to suppress symptoms. It can beeffected over a short period, be oriented over a medium term, or can bea long-term treatment, for example within the context of a maintenancetherapy.

The term “mammal” includes humans, companion animals (e.g., dogs, cats,and horses), zoo animals (e.g., zebras, elephants, and large cats),food-source animals (e.g., cows, pigs, goats, and sheep), and researchanimals (e.g., rats, mice, goats, and guinea pigs).

The present invention is directed, in part, to the discovery thatpharmaceutical compositions comprising a carbazole compound of generalstructural formulas (I) and (II) can be used to modulate NF-κB activity,such as NF-κB-mediated immune responses and conditions described inInternational Patent Application PCT/US05/25884, designating the UnitedStates, the contents of which are incorporated herein by reference.

In preferred embodiments of a carbazole compound of structural formula(I), R^(a) is methyl, ethyl, NH(CH₃), OCH₃, or forms a five-memberedaliphatic ring with R¹. In other preferred embodiments, R^(b) is methyl,ethyl, NH(CH₃), OCH₃, forms a five-membered aliphatic ring with R⁶, orforms a five-membered, nitrogen containing, aliphatic ring with R⁶. Inanother preferred embodiment, R^(d) is hydrogen, methyl, ethyl, or formsa five-membered aliphatic ring with R⁷.

In preferred embodiments, R¹ is hydrogen or forms a five-memberedaliphatic ring with R^(a). In other preferred embodiments, R² ishydrogen or hydroxy. In still other preferred embodiments, R³ ishydrogen. In further preferred embodiments, R⁴ is hydrogen. In yetfurther preferred embodiments, R⁵ is hydrogen or hydroxy. In somepreferred embodiments, R⁶ is hydrogen, forms a five-membered aliphaticring with R^(b), or forms a five-membered, nitrogen-containing aliphaticring with R^(b). In preferred embodiments, R⁷ is hydrogen or forms afive-membered ring with R^(d). In yet further preferred embodiments, nis 2 or 3.

In preferred embodiments of a carbazole compound of structural formula(II), R^(f) is methyl or ethyl, R^(g) is hydrogen, methyl, ethyl, orforms a five-membered, nitrogen containing aliphatic ring with R^(f) andR⁸, or R⁸ is hydrogen, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are hydrogen. Inyet further embodiments, p is 2 or 3.

Two additional carbazole compounds useful in the treatment of a varietyof conditions and diseases are

The present invention includes all possible stereoisomers and geometricisomers of the compounds of structural formula (I) and (II). The presentinvention includes both racemic compounds and optically active isomers.When a compound of structural formula (I) or (II) is desired as a singleenantiomer, it can be obtained either by resolution of the final productor by stereospecific synthesis from either isomerically pure startingmaterial or use of a chiral auxiliary reagent, for example, see Z. Ma etal., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution ofthe final product, an intermediate, or a starting material can beachieved by any suitable method known in the art. Additionally, insituations where tautomers of the compounds of structural formula (I) or(II) are possible, the present invention is intended to include alltautomeric forms of the compounds.

Prodrugs of compounds of structural formula (I) and (II) also can beused as the compound in a method of the present invention. It is wellestablished that a prodrug approach, wherein a compound is derivatizedinto a form suitable for formulation and/or administration, thenreleased as a drug in vivo, has been successfully employed totransiently (e.g., bioreversibly) alter the physicochemical propertiesof the compound (see, H. Bundgaard, Ed., “Design of Prodrugs,” Elsevier,Amsterdam, (1985); R. B. Silverman, “The Organic Chemistry of DrugDesign and Drug Action,” Academic Press, San Diego, chapter 8, (1992);K. M. Hillgren et al., Med. Res. Rev., 15, 83 (1995)).

Compounds of the present invention can contain one or more functionalgroups. The functional groups, if desired or necessary, can be modifiedto provide a prodrug. Suitable prodrugs include, for example, acidderivatives, such as amides and esters. It also is appreciated by thoseskilled in the art that N-oxides can be used as a prodrug.

Compounds of the invention can exist as salts. Pharmaceuticallyacceptable salts of the compounds of the invention generally arepreferred in the methods of the invention. As used herein, the term“pharmaceutically acceptable salts” refers to salts or zwitterionicforms of the compounds of structural formula (I) and (II). Salts ofcompounds of formula (I) and (II) can be prepared during the finalisolation and purification of the compounds or separately by reactingthe compound with an acid having a suitable cation. The pharmaceuticallyacceptable salts of compounds of structural formula (I) and (II) areacid addition salts formed with pharmaceutically acceptable acids.Examples of acids which can be employed to form pharmaceuticallyacceptable salts include inorganic acids such as nitric, boric,hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acidssuch as oxalic, maleic, succinic, and citric. Nonlimiting examples ofsalts of compounds of the invention include, but are not limited to, thehydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate,2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate,adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerolphsphate, hemisulfate,heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate,isethionate, salicylate, methanesulfonate, mesitylenesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate,propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate,bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate,tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzenesulphonate, and p-toluenesulfonate salts. In addition, available aminogroups present in the compounds of the invention can be quaternized withmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and steryl chlorides, bromides, and iodides; and benzyl andphenethyl bromides. In light of the foregoing, any reference tocompounds of the present invention appearing herein is intended toinclude compounds of structural formula (I) and/or (II) as well aspharmaceutically acceptable salts, hydrates, or prodrugs thereof.

The compounds of structural formula (I) and (II) also can be conjugatedor linked to auxiliary moieties that promote a beneficial property ofthe compound in a method of therapeutic use. Such conjugates can enhancedelivery of the compounds to a particular anatomical site or region ofinterest (e.g., a tumor), enable sustained therapeutic concentrations ofthe compounds in target cells, alter pharmacokinetic and pharmacodynamicproperties of the compounds, and/or improve the therapeutic index orsafety profile of the compounds. Suitable auxiliary moieties include,for example, amino acids, oligopeptides, or polypeptides, e.g.,antibodies such as monoclonal antibodies and other engineeredantibodies; and natural or synthetic ligands to receptors in targetcells or tissues. Other suitable auxiliaries include fatty acid or lipidmoieties that promote biodistribution and/or uptake of the compound bytarget cells (see, e.g., Bradley et al., Clin. Cancer Res. (2001)7:3229).

Compounds of the present invention are potent inhibitors of NF-κB. Thus,compounds of formula (I) and (II) are of interest for use in therapy,specifically for the treatment of a variety of conditions whereinhibition of NF-κB is considered beneficial. NF-κB inhibition isparticularly attractive targets because such inhibition provides effectssuch as apoptosis, antimicrobial, antiprotozoan, antiviral, andanti-inflammatory, all of which are beneficial in the treatment ofvarious disease states. The compounds of formula (I) and (II),therefore, have utility in the treatment of a number of disorders,diseases, and conditions.

The potency of a present carbazole compound is determined by measuringan ability of the compound to inhibit NF-κB activity or to activate p53.Activation of p53 typically is measured using a dose-response assay inwhich a sensitive assay system is contacted with a compound of interestover a range of concentrations, including concentrations at which no orminimal effect is observed, through higher concentrations at whichpartial effect is observed, to saturating concentrations at which amaximum effect is observed. Theoretically, such assays of thedose-response effect of activator compounds can be described as asigmoidal curve expressing a degree of activation as a function ofconcentration. The curve also theoretically passes through a point atwhich the concentration is sufficient to increase activity to a levelthat is 50% that of the difference between a baseline and the maximalactivity in the assay. This concentration is defined as the EffectiveConcentration (50%) or EC₅₀ value. Determination of an EC₅₀ value ismade using conventional biochemical (acellular) assay techniques orcell-based assay techniques.

Comparisons of the efficacy of activators often are provided withreference to comparative EC₅₀ values, wherein a higher EC₅₀ indicatesthat the test compound is less potent, and a lower EC₅₀ indicates thatthe compound is more potent, than a reference compound. Compounds of thepresent invention exhibit unexpectedly good potency, i.e., p53activation, in a luciferase reporter cell line assay. Compounds of theinvention subjected to a cell-based assay described below exhibited EC₅₀values for p53 activation of less than about 1.35 μM. In certainembodiments, compounds of the invention exhibited an EC₅₀ value of lessthan about 1.0 μM. In other embodiments, the inventive compoundsexhibited IC₅₀ values of less than about 0.75 μM, about 0.50 μM, about0.30 μM, less than about 0.20 μM, or less than 0.05 μM.

An especially important use of the present carbazole compounds is thetreatment of a cancer, an inflammation, an autoimmune disease, amicrobial, protozoan, or viral infection, a graft vs. host disease, acondition associated with HIV infection, or pre-cancerous cells whichhave acquired dependence on constitutively active NF-κB. Various cancersthat can be treated in accordance with the present invention include,but are not limited to, renal cell carcinoma, sarcoma, prostate cancer,breast cancer, pancreatic cancer, myeloma, myeloid and lymphoblasticleukemia, neuroblastoma, glioblastoma, and a cancer caused by HTLVinfection.

It is envisioned, therefore, that compounds of formula (I) and (II) areuseful in the treatment of a variety of conditions and diseases. Thus,the present invention concerns the use of compounds of formula (I) and(II), or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition containing either entity, for the manufacture of amedicament for the treatment of such conditions and diseases.

The compounds of the present invention can be therapeuticallyadministered as the neat chemical, but it is preferred to administercompounds of structural formula (I) or (II) as a pharmaceuticalcomposition or formulation. Thus, the present invention provides apharmaceutical composition comprising a compound of the formula (I) or(II) together with a pharmaceutically acceptable diluent or carriertherefor. Also provided is a process of preparing a pharmaceuticalcomposition comprising admixing a compound of formula (I) or (II) with apharmaceutically acceptable diluent or carrier therefor.

Accordingly, the present invention further provides pharmaceuticalformulations comprising a compound of structural formula (I) or (II), ora pharmaceutically acceptable salt, prodrug, or hydrate thereof,together with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic and/or prophylactic ingredients. Thecarriers are “acceptable” in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

Formulations of the present invention can be administered in a standardmanner for the treatment of the indicated diseases, such as orally,parenterally, transmucosally (e.g., sublingually or via buccaladministration), topically, transdermally, rectally, or via inhalation(e.g., nasal or deep lung inhalation). Parenteral administrationincludes, but is not limited to intravenous, intraarterial,intraperitoneal, subcutaneous, intramuscular, intrathecal, andintraarticular. Parenteral administration also can be accomplished usinga high pressure technique, like POWDERJECT™ (Powderject Pharmaceuticals,Plc, Oxford, England). The composition also can be administered in theform of an implant, which allows a slow release of the composition aswell as a slow controlled i.v. infusion.

For oral administration, including buccal administration, thecomposition can be in the form of tablets or lozenges formulated inconventional manner. For example, tablets and capsules for oraladministration can contain conventional excipients such as bindingagents (for example, syrup, acacia, gelatin, sorbitol, tragacanth,mucilage of starch, or polyvinylpyrrolidone), fillers (for example,lactose, sugar, microcrystalline cellulose, maize-starch, calciumphosphate, or sorbitol), lubricants (for example, magnesium stearate,stearic acid, talc, polyethylene glycol or silica), disintegrants (forexample, potato starch or sodium starch glycolate), or wetting agents(for example, sodium lauryl sulfate). The tablets can be coatedaccording to methods well known in the art.

Alternatively, compounds of the present invention can be incorporatedinto oral liquid preparations such as aqueous or oily suspensions,solutions, emulsions, syrups, or elixirs, for example. Moreover,formulations containing these compounds can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can contain conventional additives, forexample suspending agents, such as sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, aluminum stearategel, and hydrogenated edible fats; emulsifying agents, such as lecithin,sorbitan monooleate, or acacia; nonaqueous vehicles (which can includeedible oils), such as almond oil, fractionated coconut oil, oily esters,propylene glycol, and ethyl alcohol; and preservatives, such as methylor propyl p-hydroxybenzoate and sorbic acid.

Such preparations also can be formulated as suppositories, e.g.,containing conventional suppository bases, such as cocoa butter or otherglycerides. Compositions for inhalation typically can be provided in theform of a solution, suspension, or emulsion that can be administered asa dry powder or in the form of an aerosol using a conventionalpropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Typical topical and transdermal formulations comprise conventionalaqueous or nonaqueous vehicles, such as eye drops, creams, ointments,lotions, and pastes, or are in the form of a medicated plaster, patch,or membrane.

Additionally, compositions of the present invention can be formulatedfor parenteral administration by injection or continuous infusion.Formulations for injection can be in the form of suspensions, solutions,or emulsions in oily or aqueous vehicles, and can contain formulationagents, such as suspending, stabilizing, and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water)before use.

A composition of the present invention also can be formulated as a depotpreparation. Such long acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Accordingly, the compounds of the invention canbe formulated with suitable polymeric or hydrophobic materials (e.g., anemulsion in an acceptable oil), ion exchange resins, or as sparinglysoluble derivatives (e.g., a sparingly soluble salt).

The composition also can be formulated as a liposome preparation. Theliposome preparation can comprise liposomes which penetrate the cells ofinterest or the stratum corneum, and fuse with the cell membrane,resulting in delivery of the contents of the liposome into the cell.Liposomes are described, for example, in U.S. Pat. No. 5,077,211, U.S.Pat. No. 4,621,023, and U.S. Pat. No. 4,508,703, each incorporatedherein by reference.

For veterinary use, a compound of formula (I) or (II), or apharmaceutically acceptable salt or prodrug, is administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen androute of administration that is most appropriate for a particularanimal. Animals treatable by the present compounds and methods include,but are not limited to, pets, livestock, show animals, and zoospecimens.

Synthetic Methods

Compounds of formula (I) and (II) can be prepared by any suitable methodknown in the art, or by the following processes which form part of thepresent invention. In particular, compounds of structural formula (I)and (II) can be prepared according to the following synthetic schemes.

In the synthetic methods, the examples, and throughout thespecification, the abbreviations have the following meanings:

DMF dimethylformamide NaH sodium hydride min minutes TLC thin layerchromatography CH₂Cl₂ methylene chloride CHCl₃ chloroform MeOH methanolNa₂SO₄ sodium sulfate AlCl₃ aluminum chloride AcCl acetyl chloride LC-MSliquid chromatography-mass spectrometry Et₂O diethyl ether Na₂CO₃ sodiumcarbonate HPLC high performance liquid chromatography h hours NaHCO₃sodium bicarbonate NaCl sodium chloride HCl hydrochloric acid g gram eqequivalent mol mole mmol millimole mL milliliter H₂SO₄ sulfuric acidK₂CO₃ potassium carbonate Pd(OAc)₂ palladium acetate Pd(PPh₃)₄tetra(triphenylphosphino)palladium P(OEt)₃ triethoxyphosphine NaH sodiumhydride TfOH triflic acid EtOH ethanol NMR nuclear magnetic resonancespectrometry EtOAc ethyl acetate THF tetrahydrofuran NaOH sodiumhydroxide NMP N-methylpyn-olidinone DBU1,8-diazabicyclo[5.4.0]undec-7-ene MsCl mesyl chloride TEAtriethanolamine Na₂SO₄ sodium sulfate (Boc)₂O ditert-butyl carbonate Pypyridine PdCl₂(PPh)₃ dichloro-triphenylphosphino-palladium (II) PhNO₂nitrobenzene KOAc potassium acetate Pd(dppf)Cl₂dichloro-((bis-diphenylphosphino)ferrocenyl)-palladium(II) AcOKpotassium acetate PPh₃ triphenylphosphine PPh₃O triphenylphosphine oxideBBr₃ tribromoboron CH₃CN acetonitrile PhSH thiophenol Cs₂CO₃ cesiumcarbonate STAB sodium triacetoxyborohydride NEt₃ triethylamine DMFdimethylformamide

It should be understood that protecting groups can be utilized inaccordance with general principles of synthetic organic chemistry toprovide compounds of structural formula (I) and (II). Protectinggroup-forming reagents are well known to persons skilled in the art, forexample, see T. W. Greene et al., “Protective Groups in OrganicSynthesis, Third Edition,” John Wiley and Sons, Inc., NY, N.Y. (1999).These protecting groups are removed when necessary by appropriate basic,acidic, or hydrogenolytic conditions known to persons skilled in theart. Accordingly, compounds of structural formula (I) and (II) notspecifically exemplified herein can be prepared by persons skilled inthe art.

In addition, compounds of formula (I) and (II) can be converted to othercompounds of formula (I) and (II). Thus, for example, a particular Rsubstituent can be interconverted to prepare another suitablysubstituted compound of formula (I) or (II). Examples of appropriateinterconversions include, but are not limited to, OR^(a) to hydroxy bysuitable means (e.g., using an agent such as SnCl₂ or a palladiumcatalyst, like palladium-on-carbon), or amino to substituted amino, suchas acylamino or sulphoylamino, using standard acylating or sulfonylatingconditions.

Compounds of formula (I) and (II) can be prepared as individualstereoisomers as a racemic mixture. Individual stereoisomers of thecompounds of the invention can be prepared from racemates by resolutionusing methods known in the art for the separation of racemic mixturesinto their constituent stereoisomers, for example, using HPLC on achiral column, such as Hypersil naphthyl urea, or using separation ofsalts of stereoisomers. Compounds of the invention can be isolated inassociation with solvent molecules by crystallization from, orevaporation of, an appropriate solvent.

General Synthetic Procedures

General Procedure for Alkylation of Carbazoles

Carbazole 1 was dissolved or suspended in DMF. Then, NaH (3 eq) wasadded. The mixture was stirred at room temperature over a period of 5-10min until foaming ceased. A chloride hydrochloride (1.3 eq) was added,and the reaction mixture was kept at 50-60° C. over a period of 2-16 h(TLC monitoring; eluents: CH₂Cl₂/ethyl acetate, 1:1 for the presence ofthe starting carbazole; CHCl₃/MeOH, 9:1 for the product purity). Theresulting mixture was diluted with water. If a precipitate formed, itwas filtered off and air-dried. If no precipitate formed (Table 1), themixture was extracted with ethyl acetate. The extract was dried overNa₂SO₄, evaporated, and the residue was purified by chromatography(silica gel, CHCl₃/MeOH). The yields of products 2a and 2b are shown inTable 1.

General Procedure for Acylation of Alkylated Carbazoles

A carbazole 2 was dissolved in nitrobenzene. The solution was cooled inan ice bath, then AlCl₃ (5 eq) and AcCl (5 eq) were added. The reactionmixture was held over a period of 2-16 h (LC-MS monitoring). A sample ofthe reaction mixture was diluted with Et₂O, and the latter was decantedfrom the precipitate, then dissolved in MeOH. The resulting mixture wasdiluted with water, neutralized with Na₂CO₃, and extracted with CHCl₃.The extract was evaporated. The residue was purified first bychromatography in a short silica gel column (CHCl₃/MeOH) to remove thenitrobenzene; then, if necessary, in a silica gel column or by HPLC. Theyields of products 3a and 3b are shown in Table 1.

3,6-Diacetylcarbazole (4)

Carbazole 1 (16.9 g, 0.1 mol) was dissolved in nitrobenzene (300 mL).Anhydrous AlCl₃ (54.0 g, 0.4 mol) was added under stirring and coolingwith an ice bath. Then, AcCl (55.5 g, 0.7 mol) was added slowlydropwise. The reaction mixture was allowed to warm to room temperatureunder stirring and kept over a period of 13 h. Water (500 mL) was addedin small portions under cooling with an ice bath. The cooling bath wasremoved, and the mixture was refluxed over a period of 2 h and extractedwith CHCl₃ (3×150 mL). The combined extracts were sequentially washedwith saturated solutions of NaHCO₃ and NaCl, dried with anhydrousNa₂SO₄, and evaporated. The residue was purified by columnchromatography (silica gel, CHCl₃/MeOH) to give 12.5 g (50%) of3,6-diacetylcarbazole (4).

For the alkylation of compound 4, the general procedure for thealkylation of carbazoles was used. The yields of the products 3c-f areshown in Table 1.

Preparation of bromoalkyldiacetylcarbazoles 5a-c

Diacetylcarbazole 4 was dissolved in DMF, then NaH (3 eq) was added. Themixture was stirred over a period of 10 min at room temperature. Adibromoalkane (7 eq) was added. The reaction mixture was kept over aperiod of 1 h (5a at room temperature; 5b at 40° C.; 5c over a period of20 min at 70° C.; TLC monitoring, CH₂Cl₂/ethyl acetate, ethyl acetate4:1). The mixture then was diluted with water and extracted with ethylacetate. The combined extracts were washed with water and brine, driedwith Na₂SO₄, and evaporated. The residue was purified by columnchromatography (silica gel, CHCl₃) to give 5a (21%), 5b (29%), and 5c(74%).

Alkylation of Amines with Bromoalkyldiacetylcarbazoles 5a-c.

A bromide 5 was dissolved in DMF, the an amine was added (excess, seeTable 3). The mixture was kept at 60° C. overnight. (TLC monitoring,CH₂Cl₂/ethyl acetate, 1:1 for the presence of the starting carbazole;CHCl₃/MeOH, 9:1 for the product purity). The reaction mixture wasdiluted with water and extracted ethyl acetate. The combined extractswere dried with Na₂SO₄. The residue was purified by columnchromatography (silica gel, CHCl₃/MeOH). The product was dissolved in amixture of CH₂Cl₂ and MeOH. 4M HCl in dioxane was added, and the mixturewas evaporated. The residue was triturated with Et₂O and, if necessary,with ethyl acetate or acetone. The yields of the products 6a-h are shownin Table 3.

For the acylation of 2, a procedure similar to that described in Scheme1 was used. The yields of products 7a-d are shown in Table 4.

3,6-Bis(chloropropionyl)-9-N,N-diethylaminoethylcarbazole (8)

A solution of 1-N,N-diethylaminoethylcarbazole (0.23 g, 0.86 mmol) in 2mL of nitrobenzene was cooled in an ice bath. AlCl₃ (0.57 g, 4.3 mmol)and 3-chloropropionyl chloride (0.4 mL, 4.2 mmol) were added. Thereaction mixture was stirred overnight (LC-MS monitoring) and dilutedwith aqueous HCl. The product was extracted with CHCl₃, and the filtratewas evaporated. The residue was quickly purified by chromatography in ashort silica gel column (CHCl₃/MeOH) to give 0.38 g (91%) of thecompound 8 as its hydrochloride.

1,2,10,11-Tetrahydro-6-N,N-dimethylaminoethyl-6H-dicyclopenta[c,g]carbazole-3,9-dione(9)

Compound 2 (0.38 g, 0.79 mmol) was dissolved in 98% H₂SO₄ (3 mL). Thereaction mixture was heated to 95° C., kept at this temperature over aperiod of 2.5 h (TLC monitoring, CHCl₃/MeOH, 4:1), and poured into ice.The resulting mixture was neutralized with dry Na₂CO₃ and extracted withCHCl₃. The extract was evaporated, and the residue was purified bycolumn chromatography (CHCl₃/MeOH). The obtained crude product (0.08 g)was dissolved in MeOH. 4M HCl in dioxane was added, and the mixture wasevaporated. The residue was suspended in MeOH, and the suspension wasrefluxed. (The solid did not dissolve in the process.) The suspensionwas cooled, and the solid was filtered off to give 0.007 g (2%) ofcompound 9 as its hydrochloride.

2-Methyl-2′-nitro-1,1′-biphenyl (10a)

2-Methylphenylboronic acid (0.64 g, 4.7 mmol) and 2-nitroiodobenzene(1.0 g, 4.0 mmol) were dissolved in a mixture of MeOH (20 mL) and water(4 mL). K₂CO₃ (1.1 g, 8.0 mmol) and Pd(OAc)₂ (0.018 g, 0.08 mmol) wereadded. The reaction mixture was flushed with argon, heated to 50° C.,kept overnight at this temperature, and filtered through Celite. Thelatter was washed with MeOH. The filtrate was evaporated, and theresidue was used further without purification.

4,4′-Dimethoxy-2-nitro-1,1′-biphenyl (10h)

4-Methoxyphenylboronic acid (3.00 g, 19.7 mmol) and4-chloro-3-nitroanisole (3.69 g, 11.6 mmol) were dissolved in a mixtureof dioxane (40 mL) and water (10 mL). K₂CO₃ (5.44 g, 23.2 mmol) andPd(PPh₃)₄ (1.14 g, 0.6 mmol) were added. The reaction mixture was heatedin argon to 80° C., kept at this temperature overnight (TLC monitoring:hexane/ethyl acetate, 4:1), cooled, and filtered through Celite. Thelatter was washed with CH₂Cl₂, and the filtrate was evaporated. Theresidue was dissolved in CH₂Cl₂, and the solution was evaporated to give6.0 g of crude biphenyl 10h that was cyclized without purification.

An analogous procedure was used to obtain biphenyls 10b-g.

General Procedure for the Synthesis of Carbazoles

A crude biphenyl 10 was dissolved in (EtO)₃P. The reaction mixture waskept at 125-140° C. in a flow of argon over a period of about 48 h (TLCmonitoring: hexane/ethyl acetate, 1:1) and diluted with water. Theprecipitate was filtered off and washed with Et₂O. If no precipitateformed, the product was extracted with ethyl acetate, the extract wasevaporated, and the residue was purified in a short silica gel column(hexane/ethyl acetate). The yields of the products 11a-g are shown inTable 5.

2,7-Dimethoxy-9H-carbazole (11h)

The reaction was carried out in a vial. The crude biphenyl 10h (6.0 g)was dissolved in P(OEt)₃ (36 mL). The vial was flushed with argon. Thereaction mixture was heated to 90° C., kept at this temperatureovernight, and cooled. As a result the carbazole precipitated.Et₂O/CH₂Cl₂ mixture was added. The precipitate was filtered off andwashed with CH₂Cl₂. The filtrate was evaporated. P(OEt)₃ was addedagain, and the mixture was left for cyclization for 24 h. Theseoperations were repeated until the precipitate formation ceased, and TLCindicated that the starting biphenyl disappeared. In total 2.9 g of thecarbazole was obtained (65% calculated for two steps.)

For the alkylation of compound 11, the general procedure for thealkylation of carbazoles was used. The yields of the compounds 12a-i areshown in Table 1.

For the acylation of 12, a procedure similar to that described forScheme 1 was used. However, for the monoacetylation the amount of AcCland AlCl₃ was decreased to 1.5 eq. The yields of compounds 13a-j areshown in Table 2.

4,4,5,5-Tetramethyl-2-(4-indanone-1-yl)-[1,3,2]-dioxoborolane (14aX═CH₂)

4-Trifluoromethylsulfonyloxy-1-indanone (9.7 g, 34.6 mmol) andbis(pinacolato)diboron (11.4 g, 45.0 mmol) were dissolved in dioxane(100 mL). AcOK (6.8 g, 69.2 mmol) and Pd(dppf)₂Cl₂ (1.3 g, 1.8 mmol)were added. The reaction mixture was heated in a flow of argon to 80°C., kept at this temperature overnight, cooled, and filtered throughCelite. The filtrate was evaporated. The residue was dissolved in CH₂Cl₂and purified in a short silica gel column (hexane/ethyl acetate) to give9.5 g of a product containing 20% (mass) of bis(pinacolato)diboron. Theproduct was used for the next step without additional purification.

The boronic ester from the bromide was prepared in the same way. If 1 eqof bis(pinacolato)diboron was used, a more pure product was obtained.

4,4,5,5-Tetramethyl-2-[4-(2-methylisoindolin-1-one)-yl]-[1,3,2]-dioxoborolane(14b X═NMe)

4-Bromo-2-methylisoindolin-1-one (3.23 g, 14.3 mmol) andbis(pinacolato)diboron (4.72 g, 18.6 mmol) were dissolved in dioxane (60mL). AcOK (2.80 g, 28.6 mmol) and Pd(dppf)₂Cl₂ (0.5 g, 0.7 mmol) wereadded. The reaction mixture was heated in a flow of argon to 80° C.,kept at this temperature overnight, cooled, and filtered through Celite.The filtrate was evaporated. The residue was dissolved in CH₂Cl₂ andpurified in a short silica gel column (hexane/ethyl acetate) to give 4.2g of a product containing 20% (mass) of bis(pinacolato)diboron. Theproduct was used for the next step without additional purification.

Biphenyl 15a (X═CH₂, R═H)

4,4,5,5-Tetramethyl-2-(4-indanone-1-yl)-[1,3,2]-dioxoborolane (2.17 g,8.4 mmol) and o-nitroiodobenzene (2.70 g, 10.9 mmol) were dissolved in amixture of dioxane (30 mL) and water (5 mL). K₂CO₃ (2.30 g, 16.7 mmol)and Pd(PPh₃)₄ (0.48 g, 0.4 mmol) were added. The reaction mixture washeated in a flow of argon to 80° C., kept at this temperature over aperiod of 24 h (TLC monitoring: hexane/ethyl acetate, 4:1), cooled, andfiltered through Celite. The filtrate was evaporated. The residue wasdissolved in CH₂Cl₂. An undissolved precipitate was filtered off. Thefiltrate was partially evaporated, and the product was purified in ashort silica gel column (hexane/ethyl acetate) to give 2.5 g of aproduct containing PPh₃O. The product was cyclized without additionalpurification.

Biphenyl 15b (X═CH₂, R═OMe)

4,4,5,5-Tetramethyl-2-(4-indanone-1-yl)-[1,3,2]-dioxoborolane (1.20 g,4.6 mmol) and o-nitroiodobenzene (0.87 g, 4.6 mmol) were dissolved in amixture of dioxane (10 mL) and water (2 mL). K₂CO₃ (1.28 g, 9.2 mmol)and Pd(PPh₃)₄ (0.27 g, 0.2 mmol) were added. The reaction mixture washeated in a flow of argon to 80° C., kept at this temperature over aperiod of 24 h (TLC monitoring: hexane/ethyl acetate, 4:1), cooled, andfiltered through Celite. The filtrate was evaporated. The residue wasdissolved in CH₂Cl₂. An undissolved precipitate was filtered off. Thefiltrate was partially evaporated, and the product was purified in ashort silica gel column (hexane/ethyl acetate) to give 1.25 g of aproduct containing PPh₃O. The product was cyclized without additionalpurification.

Biphenyl 15c (X═NMe, R═H)

4,4,5,5-Tetramethyl-2-[4-(2-methylisoindolin-1-one)-yl]-[1,3,2]-dioxoborolane(2.43 g, 8.9 mmol) and o-nitroiodobenzene (2.44 g, 9.80 mmol) weredissolved in a mixture of dioxane (30 mL) and water (6 mL). K₂CO₃ (2.50g, 18.1 mmol) and Pd(PPh₃)₄ (0.51 g, 0.4 mmol) were added. The reactionmixture was heated in a flow of argon to 80° C., kept at thistemperature over a period of 24 h (TLC monitoring: hexane/ethyl acetate,4:1), cooled, and filtered through Celite. The filtrate was evaporated.The residue was dissolved in CH₂Cl₂. An undissolved precipitate wasfiltered off. The filtrate was partially evaporated, and the product waspurified in a short silica gel column (hexane/ethyl acetate) to give 2.3g of a product containing PPh₃O. The product was cyclized withoutadditional purification.

Biphenyl 15d (X═NMe, R═OMe)

4,4,5,5-Tetramethyl-2-[4-(2-methylisoindolin-1-one)-yl]-[1,3,2]-dioxoborolane(0.97 g, 3.6 mmol) and 4-chloro-3-nitroanisole (0.67 g, 3.6 mmol) weredissolved in a mixture of dioxane (10 mL) and water (2 mL). K₂CO₃ (0.98g, 7.2 mmol) and Pd(PPh₃)₄ (0.21 g, 0.2 mmol) were added. The reactionmixture was heated in a flow of argon to 80° C., kept at thistemperature over a period of 24 h (TLC monitoring: hexane/ethyl acetate,4:1), cooled, and filtered through Celite. The filtrate was evaporated.The residue was dissolved in CH₂Cl₂. An undissolved precipitate wasfiltered off. The filtrate was partially evaporated, and the product waspurified in a short silica gel column (hexane/ethyl acetate) to give0.76 g of a product containing PPh₃O. The product was cyclized withoutadditional purification.

Carbazole 16a (X═CH₂, R═H)

The reaction was carried out in a vial. 4-(2-Nirophenyl)indanone-1 (2.54g, 10.0 mmol) was dissolved in P(OEt)₃ (8 mL). The vial was flushed withargon. The reaction mixture was heated to 90° C., kept at thistemperature overnight, and cooled. As a result the carbazoleprecipitated. CH₂Cl₂ was added. The precipitate was filtered off andwashed with CH₂Cl₂. The filtrate was evaporated. P(OEt)₃ (2 mL) wasadded again, and the mixture was left for cyclization for 24 h. Theseoperations were repeated until the precipitate formation ceased, and TLCindicated that the starting biphenyl disappeared. In total 0.58 g of thecarbazole was obtained.

Carbazole 16b (X═CH₂, R═OMe)

The reaction was carried out in a vial.4-(4-Methoxy-2-nitrophenyl)-indanone-1 (1.25 g, 4.4 mmol) was dissolvedin P(OEt)₃ (8 mL). The vial was flushed with argon. The reaction mixturewas heated to 90° C., kept at this temperature overnight, and cooled. Asa result the carbazole precipitated. CH₂Cl₂ was added. The precipitatewas filtered off and washed with CH₂Cl₂. The filtrate was evaporated.P(OEt)₃ (1 mL) was added again, and the mixture was left for cyclizationfor 24 h. These operations were repeated until the precipitate formationceased, and TLC indicated that the starting biphenyl disappeared. Intotal 0.39 g of the carbazole was obtained.

Carbazole 16c (X═NMe, R═H)

The reaction was carried out in a vial.4-(2-Nitrophenyl)-2-methylisoinolin-1-one (2.29 g, 8.5 mmol) wasdissolved in P(OEt)₃ (10 mL). The vial was flushed with argon. Thereaction mixture was heated to 90° C., kept at this temperatureovernight, and cooled. As a result the carbazole precipitated. CH₂Cl₂was added. The precipitate was filtered off and washed with CH₂Cl₂. Thefiltrate was evaporated. P(OEt)₃ (0.5 mL) was added again, and themixture was left for cyclization for 24 h. These operations wererepeated until precipitate formation ceased, and TLC indicated that thestarting biphenyl disappeared. In total 0.4 g of the carbazole wasobtained.

Carbazole 16d (X═NMe, R═OMe)

The reaction was carried out in a vial.4-(4-Methoxy-2-nitrophenyl)-2-methylisoinolin-1-one (0.76 g, 2.6 mmol)was dissolved in P(OEt)₃ (6 mL). The vial was flushed with argon. Thereaction mixture was heated to 90° C., kept at this temperatureovernight, and cooled. As a result the carbazole precipitated. CH₂Cl₂was added. The precipitate was filtered off and washed with CH₂Cl₂. Thefiltrate was evaporated. P(OEt)₃ (0.5 mL) was added again, and themixture was left for cyclization for 24 h. These operations wererepeated until precipitate formation ceased, and TLC indicated that thestarting biphenyl disappeared. In total, 0.34 g of the carbazole wasobtained.

For the alkylation of 16, the general procedure for the alkylation ofcarbazoles was used. The yields of the products 17a-f are shown in Table1.

For the acylation of 17, a procedure similar to that described forScheme 1 was used. The yields of the products 18a-d are shown in Table2.

General Procedure for Dimethylation.

A methoxy compound was dissolved in CH₂Cl₂. The solution was cooled to−40° C. A 0.5M solution of BBr₃ in DCM (4 eq, for one methoxy group) wasadded in a flow of argon. After 10 min the cooling bath was removed. Thereaction mixture was heated to room temperature, kept over a period of 1h (TLC monitoring, CHCl₃/MeOH, 4:1), and poured into a mixture ofaqueous NaHCO₃ and CH₂Cl₂. The organic layer was separated, and theaqueous one was extracted once more with CH₂Cl₂. The combined extractswere dried with Na₂SO₄ and evaporated. The product was purified bycolumn chromatography (CHCl₃/MeOH). The yields of the products 19a-h areshown in Table 6.

2-Hydroxy-9-N,N-diethylaminoethylcarbazole (20)

2-Methoxy-9-N,N-diethylaminoethylcarbazole 12 g was dissolved in CH₂Cl₂(10 mL). The solution was cooled to −40° C. A 0.5M solution of BBr₃ DCM(6 mL, 3.00 mmol) was added in a flow of argon. As a result an orangesuspension formed. The reaction mixture was heated to room temperature,kept over a period of 1.5 h, and poured into a mixture of aqueous NaHCO₃and CH₂Cl₂. The organic layer was separated, and the aqueous layer wasextracted once more with CH₂Cl₂. The combined extracts were dried withNa₂SO₄ and evaporated. The product was purified by column chromatography(CHCl₃/MeOH) to give 0.176 g (92%) of the product.

2-Acetoxy-9-N,N-diethylaminoethylcarbazole (21)

A solution of compound 20 (0.176 g, 0.62 mmol) in Ac₂O (2 mL) wasrefluxed over a period of 30 min and poured into water. The resultingmixture was neutralized with NaHCO₃ and extracted ethyl acetate. Theextract was evaporated to give 0.16 g (79%) of the product.

3-Acetyl-2-hydroxy-9-N,N-diethylaminoethylcarbazole (22)

Compound 21 (0.16 g, 0.49 mmol) was dissolved in PhNO₂ (2 mL), and AlCl₃(0.1 g, 0.75 mmol) was added. The reaction mixture was heated in an oilbath to 100° C., kept at this temperature over a period of 2 h, dilutedwith water, neutralized with Na₂CO₃, and extracted with HCl₃. Theextract was evaporated. The residue was purified by chromatography in ashort silica gel column (CHCl₃/MeOH) to give 0.044 g (28%) of compound22.

TABLE 1 Alkylation of carbazoles Yield 2a

  100% 2b

  100% 12a

   47% 12b

   98%^(a) 12c

   59%^(a) 12d

 ~25%^(b) 12e

   39%^(c) 12f

   91% 12g

   91%^(a) 17a

   31% 17b

   28% 12h^(d)

  100% 17c

   67% 12i^(d)

  100% 17d

   42% 17e

   32% 3c

   64% 3d

   78% 3e

   80% 3f

   73% ^(a)Contains DMF. ^(b)The yield is not precise. Crude carbazolewas used for the preparation. Purification via hydrochloride.^(c)Purification via hydrochloride. ^(d)Isolated as crystallinesubstances immediately after diluting the reaction mixture with water.

TABLE 2 Acetylation of alkylated carbazoles Yield 3a

57% 3b

64% 13a

55% 13b

31% 13c

63% 13d

92% 13e

88% 13f

95% 13g

69% 13h

57%^(a) 18a

33% 13i

74% 18b

b 13j

48% 18c

46%^(a) 18d

81% ^(a)After HPLC Purification. ^(b)A mixture with the startingcompound was isolated and was used further without separation.

TABLE 3 Alkylation of carbazoles with bromoalkyldiacetylcarbazoles Yield6a

78% 6b

96% 6c

73% 6d

77% 6e

41% 6f

54% 6g

54% 6h

46%

TABLE 4 Yield 7a

32% 7b

54% 7c

14% ^(a) 7d

64% ^(a) After HPLC purification

TABLE 5 Boronic Yield (calculated acid Halide Biphenyl Carbazole for thehalide) 11a

35% 11b^(a)

39% 11c

82% 11d

^(b) 11e

37% 11f

16%^(c) 11g^(a)

48% ^(a)Precipitated as crystals. ^(b)The product contained (EtO)₃PO andwas used without purification. ^(c)Only the yield of the targetregioisomer is given.

TABLE 6 Yield 19a

43% 19b

23%^(a) 19c

29%^(b) 19d

34%^(b) 19e

10%^(a) 19f

23%^(c) ^(a)After HPLC purification. ^(b)Obtained after working with amixture separated by HPLC. ^(c)Purification via hydrochloride.

EXAMPLES Example 1

[3-(9H-carbazol-9-yl)propyl]dimethylamine (30)

Carbazole 3.0 g (18.0 mmol) was dissolved in DMF (20 mL). Then, 60% NaHin paraffin (2.5 g, 62.5 mmol) was added, and the mixture was stirredfor 10 min. 2-N,N-dimethylaminopropyl chloride 3.0 g (19.0 mmol) wasadded in portions, whereupon the temperature rose to 45-50° C. Thereaction mixture was kept at this temperature for 2.5 h (TLC monitoring,CHCl₃/MeOH, 9:1). The obtained mass was carefully poured into ice/watermixture and extracted with ethyl acetate. The extract was dried withNa₂SO₄ and evaporated to give compound 30 (4.8 g, 100%) as a brown fluidoil.

1,1′-{9-[3-(dimethylamino)propyl]-9H-carbazole-3,6-diyl}bis(2-methylpropan-1-one)(Example 1)

Compound 30 (0.25 g, 1.0 mmol) was dissolved in nitrobenzene (5 mL).AlCl₃ (0.6 g, 4.5 mmol) and then isobutyroyl chloride (0.6 mL, 5.7 mmol)were added in portions. The reaction mixture was stirred for 40 min(LC/MS monitoring). The obtained mixture was poured into ice/watermixture and extracted with CHCl₃. The combined extracts were evaporated,and the residue was purified by chromatography in a short thick silicagel column (eluent: CHCl₃/MeOH 99:1→90:10) to afford 0.189 g (47%) ofthe product. ¹H NMR (DMSO-d₆): δ 1.20 (12H, d, J=6.7 Hz); 1.90-1.97 (2H,m); 2.11 (6H, s); 2.18 (2H, t, J=6.7 Hz); 3.91 (2H, septet, J=6.7 Hz),4.50 (2H, t, J=6.6 Hz); 7.76 (2H, d, J=8.7 Hz); 8.14 (2H, dd, J=8.7 Hz,J=1.5 Hz); 9.11 (2H, d, J=1.5 Hz). ELI-MS: 100%, ESI-MS: m/z 392 [M+H]⁺.

Example 2

1,1′-(9H-Carbazole-3,6-diyl)diethanone (31)

Carbazole (16.9 g, 0.1 mol) was dissolved in nitrobenzene (300 mL).Anhydrous AlCl₃ (54.0 g, 0.4 mol) was added under stirring in an icebath. Then AcCl (55.5 g, 0.7 mol) was added slowly dropwise. Thereaction mixture was allowed to warm to room temperature under stirringand kept for 13 h. Water (500 mL) was added in small portions undercooling in an ice bath in order to avoid violent foaming. The coolingbath was removed, and the mixture was refluxed with a condenser for 2 h.The product was extracted with chloroform (3×150 mL). The combinedextracts were sequentially washed with saturated solutions of NaHCO₃ andNaCl, dried with anhydrous Na₂SO₄, and evaporated. The residue waspurified by column chromatography (silica gel, CHCl₃-MeOH) to afford 112.5 g (50%).

1,1′-{9-[2-(1-Methylpyrrolidin-2-yl)ethyl]-9H-carbazole-3,6-diyl}diethanone(Example 2, 3c)

The diacetyl derivative 31 (0.97 g, 3.86 mmol) was dissolved in DMF (7mL). NaH (0.54 g, 13.5 mmol) was added, and the mixture was stirred for3-5 min at room temperature. 2-(2-Chloroethyl)-1-methylpyrrolidinehydrochloride (1.07 g, 5.8 mmol) was added. The reaction mixture wasstirred for 24 h at 60° C. (TLC monitoring, CHCl₃/MeOH, 9:1), dilutedwith water, and extracted with ethyl acetate. The extract was dried withNa₂SO₄ and evaporated. The residue was purified by column chromatography(silica gel, CHCl₃/MeOH 99:1→:10) to give 0.90 g (64%) of the product.¹H NMR (DMSO-d₆): δ 1.41-1.49 (1H, m); 1.54-1.73 (3H, m); 1.76-1.85 (1H,m); 1.97-2.13 (3H, m); 2.14 (3H, s); 2.70 (6H, s); 2.86-2.93 (1H, m);4.46-4.50 (2H, m); 7.72 (2H, d, J=8.8 Hz); 8.12 (2H, dd, J=8.8 Hz, J=1.6Hz); 9.04 (2H, d, J=1.6 Hz). ELSD: 100%, ESI-MS: m/z 363 [M+H]⁺.

Example 3

[2-(9H-Carbazol-9-yl)ethyl]diethylamine (32)

Carbazole (10.0 g, 59.8 mmol) was dissolved in DMF (60 mL). Then, 60%NaH in paraffin (7.2 g, 180.0 mmol) was added in portions. The mixturewas stirred for 10 min. 2-N,N-Diethylaminoethyl chloride hydrochloride(10.5 g, 61.0 mmol) was added in portions, whereupon the temperaturerose to 50° C. The reaction mixture was kept at this temperature for 2.5h (TLC monitoring, hexane/ethyl acetate 4:1). The resulting mass wascarefully poured into ice/water mixture and extracted with ethylacetate. The extract was dried with Na₂SO₄ and evaporated to givecompound 1 (16.0 g, 100%) as a fluid brown oil.

1,1′-{9-[2-(Diethylamino)ethyl]-9H-carbazole-3,6-diyl}bis(3-chloropropan-1-one)hydrochloride (33)

A solution of compound 32 (17.9 g, 67.3 mmol) in nitrobenzene (150 mL)was cooled in an ice bath. AlCl₃ (45.0 g, 337.1 mmol) was added inportions. Then 3-chloropropionyl chloride (32.4 mL, 336.7 mmol) wasadded dropwise for 10 min. The reaction mixture was stirred for 40 min(LC/MS monitoring), poured into a mixture of ice with diluted HCl, andextracted with CHCl₃. The extract was evaporated. The residue waspurified by chromatography in a thick short column (silica gel,CHCl₃/MeOH 99:1→90:10) to afford 22.9 g (76.3%) of the hydrochloride 33.

6-[2-(Diethylamino)ethyl]-10,11-dihydro-1H-dicyclopenta[c,g]carbazole-3,9(2H,6H)-dionehydrochloride (Example 3)

The hydrochloride 2 (22.9 g, 51.3 mmol) was dissolved in 98% H₂SO₄ (150mL). The solution was kept at 80° C. for 4 h (TLC monitoring, CHCl₃/MeOH4:1) and poured into ice. The resulting mixture was neutralized withNa₂CO₃ and extracted with CHCl₃. The extract was evaporated. The residuewas purified by chromatography in a short thick column (silica gel,CHCl₃/MeOH 99:1→90:10) and recrystallized from MeOH to give 0.562 g (3%)of the product. The latter was dissolved in CH₂Cl₂, HCl in dioxane wasadded, and the mixture was evaporated to dryness. The residue was washedwith ether and dried to give 0.6358 g the hydrochloride. ¹H NMR(DMSO-d₆): δ 1.26 (6H, t, J=7.4 Hz); 2.77-2.80 (4H, m); 3.42-3.47 (2H,m); 3.23-3.34 (4H, m); 3.83-3.85 (4H, m); 5.88 (2H, t, J=7.8 Hz); 7.84(2H, d, J=8.6 Hz); 7.97 (2H, d, J=8.6 Hz); 10.75 (1H, br.s). ELSD: 100%,ESI-MS: m/z 375 [M+H]⁺.

Example 4

4-(4,4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)indan-1-one (34)

Potassium acetate (17.8 g, 181.6 mmol) and Pd(dppf)Cl₂ (3.3 g, 4.5 mmol)were added to a solution of 4-bromo-1-indanone (19.3 g, 91.5 mmol) andbis(pinacolato)diboron (23.2 g, 91.3 mmol) in dioxane (300 mL). Themixture was heated to 80° C. under argon, kept at this temperature for16 h, cooled, filtered through Celite, and evaporated. The residue wasdissolved in CH₂Cl₂. The product was purified on a short thick columnwith silica gel (eluent: hexane-ethyl acetate 100:0→50:50). Yield of 34:23.4 g. The product containing bis(pinacolato)diboron (about 7 molar %)was used in the next step without additional purification.

4-(4-Methoxy-2-nitrophenyl)indan-1-one (35)

Potash (7.5 g, 54.3 mmol) and Pd(PPh₃)₄ (1.6 g, 1.4 mmol) were added toa solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indan-1-one34 (7.0 g, 27.1 mmol) and 4-chloro-3-nitroanisole (5.1 g, 27.1 mmol) ina mixture of dioxane (80 mL) and water (20 mL). The mixture obtained washeated to 80° C. under argon, kept at this temperature for 16 h (TLCmonitoring, eluent: hexane-ethyl acetate, 1:1), cooled, filtered throughCelite, evaporated, and dissolved in CH₂Cl₂. Then the mixture wasfiltered off from the insoluble portion, evaporated with silica gel, andpurified on a short thick column with silica gel (eluent: hexane-CH₂Cl₂100:0→0:100, CH₂Cl₂-ethyl acetate 100:0→50:50). Yield of 35: 5.59 g. Theproduct was used in the next step without additional purification.

8-Methoxy-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one (36)

Biphenyl 35 obtained (5.59 g) was divided into five portions (1.11 geach); then P(OEt)₃ (per 7 mL) was added to each portion. The resultingmixture was subjected to argon blow in a flask, heated up to 90° C.,kept at this temperature for 3 days, and cooled down. Carbazole wasprecipitated. Then the reaction mixture was diluted with ether, theprecipitate was filtered off and washed with CH₂Cl₂. If the initialbiphenyl remained in the filtrate (TLC monitoring, eluent: hexane-ethylacetate, 1:1), this filtrate was evaporated and P(OEt)₃ (per 1 mL intoeach flask) was added. The mixture was subjected to repeated cyclizationfor a day. The procedure repeated until the precipitation ceased and TLCdata showed the absence of the initial biphenyl. Total yield ofcarbazole 36: 2.16 g.

6-[3-(Dimethylamino)propyl]-8-methoxy-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(37)

Compound 36 (1.0 g, 3.98 mmol) was suspended in CH₂Cl₂ (10 mL); NaH(0.75 g, 12.0 mmol, 3 eq.) was added. The mixture was stirred at roomtemperature for 5-10 min; then 3-dimethylamino-1-propyl chloridehydrochloride (0.75 g, 4.74 mmol, 1.2 eq.) was added. The reactionmixture was heated to 70° C., kept at this temperature for 2 h (TLCmonitoring, eluent: CH₂Cl₂-ethyl acetate, 1:1—the presence of initialcarbazole, CHCl₃-MeOH, 9:1—purity of the product). The resulting mixturewas diluted with water, extracted with ethyl acetate, evaporated, andpurified on a short thick column with silica gel, eluent: CHCl₃-MeOH99:1→90:10. Yield of 37: 0.84 g (63%).

9-Acetyl-6-[3-(dimethylamino)propyl]-8-methoxy-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(38)

A solution of compound 37 (0.84 g, 2.51 mmol) in PhNO₂ (20 mL) wascooled in an ice bath. Then AlCl₃ (1.7 g, 12.7 mmol, 5 eq.) and afterthat, AcCl (0.9 mL, 12.7 mmol, 5 eq.) were added. The mixture was keptfor 40 min (LC/MS monitoring), diluted with water, neutralized withNa₂CO₃, extracted with CHCl₃, and evaporated. The product was purifiedon a short thick column, eluent: CHCl₃-MeOH 99:1→90:10. Yield of 38:0.658 g (69%).

9-Acetyl-6-[3-(dimethylamino)propyl]-8-hydroxy-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(Example 4; 19c)

A 0.5 M solution of BBr₃ (16.5 mL, 5 eq.) was added dropwise under argonto a solution of compound 38 (0.658 g, 1.74 mmol) in CH₂Cl₂ (100 mL)cooled to −40° C. The mixture was kept for 10 min; then a cooling bathwas removed and the mixture was heated to room temperature and kept for1-2 h (TLC monitoring, eluent: CHCl₃/NH₃-MeOH, 9:1). The resultingmixture was poured in a mixture of aqueous NaHCO₃ solution and CH₂Cl₂.The organic layer was separated; the aqueous one was extracted withCH₂Cl₂, dried over Na₂SO₄, evaporated, and dissolved in a mixture:CHCl₃-MeOH-water, 40:9:1. The product was purified in a silica gelcolumn with the use of the latter mixture as an eluent. Yield of Example4: 0.258 g (41%). For the preparation of hydrochloride, the productisolated was dissolved in a mixture: CH₂Cl₂-MeOH; a solution of HCl indioxane was added. The resulting mixture was evaporated to dryness; theresidue was washed with ether.

¹H NMR spectrum (DMSO-d₆): δ 2.14-2.20 (2H, m); 2.70 (6H, d, J=4.9 Hz);2.78-2.81 (2H, m); 3.12-3.17 (2H, m); 3.60-3.62 (2H, m); 4.51 (2H, t,J=7.1 Hz); 7.28 (1H, s); 7.73 (2H, s—degenerated AB system); 8.55 (1H,s); 10.40 (1H, br. s); 12.76 (1H, s). ELSD: 100%, ESI-MS: m/z 364[M+H].⁺

Example 5

4,4′-dimethoxy-2-nitrobiphenyl (39)

Potash (9.10 g, 65.9 mmol) and Pd(PPh₃)₄ (1.90 g, 1.6 mmol) were addedto a solution of 4-methoxyphenylboronic acid (5.0 g, 32.9 mmol) and4-chloro-3-nitroanizole (6.17 g, 32.9 mmol) in a mixture of dioxane (80mL) and water (20 mL). The resulting mixture was heated to 80° C. underargon, kept at this temperature for 16 h (TLC monitoring, eluent:hexane-ethyl acetate, 4:1), cooled, and filtered through Celite. Theproduct was washed off with CH₂Cl₂. The residue was dissolved in CH₂Cl₂and purified on a short thick column (eluent: hexane-CH₂Cl₂100:0→0:100,CH₂Cl₂-ethyl acetate 100:0→50:50). Yield of 1: 8.47 g.

2,7-dimethoxy-9H-carbazole (40)

Compound 39 (8.47 g) was divided into eight portions (1.06 g each).Then, P(OEt)₃ (per 7 mL) was added to each portion. The resultingmixtures were subjected to argon blow, heated to 90° C., kept at thistemperature for 3 days, cooled, and diluted with ether. The precipitateobtained was filtered and washed with CH₂Cl₂. Yield of carbazole 40:3.33 g.

[3-(2,7-dimethoxy-9H-carbazol-9-yl)propyl]dimethylamine (40)

Compound 40 (0.7 g, 3.1 mmol) was suspended in DMF (6 mL). Sodiumhydride (0.4 g, 10.0 mmol) was added and the mixture was stirred at roomtemperature for 5-10 min. Then 3-dimethylamino-1-propyl chloridehydrochloride (0.73 g, 4.6 mmol) was added. The mixture obtained washeated to 50-60° C., kept at this temperature for 16 h (TLC monitoring,eluent: CH₂Cl₂-ethyl acetate, 1:1—the presence of the initial carbazole;CH₂Cl₂-MeOH, 9:1—the purity of the product), and diluted with water. Thewhite precipitate obtained was left to settle for 1 h, filtered off, anddried in air. Yield of 41: 0.96 g (100%).

1,1′-{9-[3-(dimethylamino)propyl]-2,7-dimethoxy-9H-carbazole-3,6-diyl}diethanone(42)

Compound 41 (0.96 g, 3.2 mmol) was dissolved in PhNO₂ (20 mL); thesolution was cooled on an ice bath. Then, AcCl (1.1 mL, 15.4 mmol) wasadded, followed by AlCl₃ (2.1 g, 15.7 mmol) in portions. The resultingmixture was kept for 40 min (LC/MS monitoring), diluted with water,neutralized with Na₂CO₃, extracted with CHCl₃, and evaporated. Theproduct was purified on a short thick column with silica gel. Yield of42: 0.787 g (62%).

1,1′-{9-[3-(dimethylamino)propyl]-2,7-dihydroxy-9H-carbazole-3,6-diyl}diethanone(Example 5; 19b)

A 0.5 M solution of BBr₃ (22.5 mL) was added dropwise under argon to asolution of compound 42 (0.787 g, 1.99 mmol) in CH₂Cl₂ (90 mL), cooledto −40° C. After 10 min, a cooling bath was removed; the mixture washeated up to room temperature and kept for 1-2 h (TLC monitoring,eluent: chloroform-methanol, 4:1). The resulting mixture was poured in amixture of aqueous soda solution and CH₂Cl₂. The organic layer wasseparated. The aqueous layer was extracted with CH₂Cl₂, dried overNa₂SO₄, evaporated, and dissolved in a mixture CHCl₃-MeOH-water, 40:9:1.The product was purified on a column. Yield of Example 5: 0.379 g (52%).

Then the product was dissolved in a mixture CH₂Cl₂-MeOH. A solution ofHCl in dioxane was added. The resulting solution was evaporated todryness; the residue was washed with CH₃CN and ether. From a base formof the product (0.379 g) 0.339 g of the hydrochloride was isolated.

¹H NMR (DMSO-d₆) δ: 2.09-2.13 (2H, m); 2.72 (6H, s); 2.77 (6H, s);3.13-3.16 (2H, m); 4.34 (2H, t, J=7.1 Hz); 7.13 (2H, s); 8.84 (2H, s);10.17 (1H, br. s); 12.82 (2H, s). ELSD: 100%, ESI-MS: m/z 369 [M+H]⁺.

Example 6

Potash (8.1 g, 58.7 mmol) and Pd(PPh₃)₄ (1.7 g, 1.5 mmol) were added toa solution of compound 44 (7.51 g, 29.1 mmol) and4-chloro-3-nitrobenzene (4.6 g, 29.1 mmol) in a mixture of dioxane (80mL) and water (20 mL). The mixture obtained was heated under argon to80° C., kept at this temperature for 16 h (TLC monitoring, eluent:hexane-ethyl acetate, 1:1), cooled, filtered through Celite, andevaporated. The resulting product was dissolved in CH₂Cl₂ and aninsoluble residue was filtered off. The resulting product was evaporatedwith silica gel and purified on a short thick column (eluent:hexane-CH₂Cl₂100:0→0:100, CH₂Cl₂-ethyl acetate 100:0→50:50). Compound 45(5.0 g) was isolated and used in the next step without purification.

1,6-Dihydrocyclopenta[c]carbazol-3(2H-one (46)

Compound 45 (5.0 g) was divided into five portions (1.0 g each). P(OEt)₃(per 7 mL) was added to each portion. The resulting product wassubjected to argon blow in a flask, heated to 90° C., kept at thistemperature for 3 days, then cooled. The carbazole precipitate wasdiluted with ether. The precipitate was filtered and washed with ether.If the initial biphenyl was present in the filtrate (TLC monitoring,eluent: hexane-ethyl acetate, 1:1), this filtrate was evaporated, thenP(OEt)₃ was added (1 mL to each flask), and subjected to cyclization for1 day. The procedure was repeated until the precipitation ceased and TLCshowed the absence of the initial biphenyl. Carbazol 46 (2.7 g) wasobtained.

2-Nitro-N-propylbenzenesulfonamide (49)

A solution of 2-nitrophenylsulfochloride (8 g, 36 mmol) in ethyl acetate(25 mL) was added dropwise at room temperature to a mixture ofpropylamine (3 mL, 36.0 mmol), ethyl acetate (15 mL), Na₂CO₃ (4 g, 37.7mmol), and water (25 mL). The resulting solution was stirred for 2 h(TLC monitoring, eluent: CH₂Cl₂). The organic layer was separated,washed with water, a citric acid solution, dried over Na₂SO₄, andevaporated. The precipitate crystallized as a white mass, which waswashed off with hexane. Yield of 49: (7.47 g, 85%).

N-(3-Bromopropyl)-2-nitro-N-propylbenzenesulfonamide (50)

1,3-Dibromopropane (8.5 mL, 82.0 mmol, 10 eq.) was added to a solutionof compound 49 (2.0 g, 8.2 mmol) in DMF (20 mL). Then NaH (0.6 g, 15.0mmol) was added in portions. The temperature rose to 50-60° C. Themixture was stirred at this temperature for 20 min (TLC monitoring,eluent: CH₂Cl₂). Then the mixture was carefully poured into ice. Theproduct was extracted with ethyl acetate, and the extract wasevaporated. The residue was washed from 1,3-dibromopropane with hexane.The resulting product was dissolved in CH₂Cl₂ and purified on a shortthick column with silica gel, eluent: hexane-CH₂Cl₂ 100:0→0:100.Compound 50 (1.87 g, 62%) was isolated as rapidly crystallizable oil.

2-Nitro-N-[3-(3-oxo-2,3-dihydrocyclopenta[c]carbazol-6(1H)-yl)propyl]-N-propylbenzenesulfonamide(47)

Compound 46 (0.538 g, 2.43 mmol) and bromide 50 (0.9 g, 2.46 mmol) weredissolved in CH₂Cl₂ (30 mL). Then NaH (0.15 g, 3.75 mmol, 1.5 eq.) wasadded. The reaction mixture was stirred for 20 min (TLC monitoring,eluent: hexane-ethyl acetate, 1:1). Then, the mixture was poured ontoice. The product was extracted with ethyl acetate, and the extract wasevaporated in a rotary evaporator. The residue of CH₂Cl₂ was removed inhigh vacuum. Methanol was added to the hardened precipitate. The productwas triturated; the precipitate was filtered off. Yield of 47: 0.638 g(52%).

N-[3-(9-Acetyl-3-oxo-2,3-dihydrocyclopenta[c]carbazol-6(1H)-yl)propyl]-2-nitro-N-propylbenzenesulfonamide(48)

Compound 47 (0.638 g, 1.26 mmol) was dissolved in PhNO₂ (7 mL). Thesolution was cooled on an ice bath, AlCl₃ (0.67 g, 5.02 mmol) and thenAcCl (0.45 mL, 6.31 mmol) were added. The mixture was kept for 40 min(LC/MS monitoring), diluted with water. The product was extracted withCH₂Cl₂. The extract was evaporated. The residue was evaporated, and theproduct was passed through Celite, eluent: CH₂Cl₂-ethyl acetate100:0→50:50. Yield of 48: 0.453 g (66%).

9-Acetyl-6-[3-(propylamino)propyl]-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(Example 6)

Compound 48 (0.496 g, 0.91 mmol) was suspended in a mixture of MeOH (10mL) and CHCl₃ (15 mL). The mixture was heated to boiling. Then Cs₂CO₃(0.6 g, 1.82 mmol) was added and at once PhSH (0.2 mL, 1.96 mmol) waspoured (the solution turned blue). The resulting solution was refluxedfor 2 h (TLC monitoring, eluent: CHCl₃-MeOH, 9:1). During this periodthe reaction mixture turned green. Then, the mixture was evaporated todryness. A dilute solution of citric acid was added. A yellowprecipitate was filtered off, washed with ether, suspended in CH₃CN, andrefluxed. After cooling, the beige precipitate was filtered off from theyellow filtrate, and then washed with ether. The resulting product(0.251 g, 76%) was isolated. For the transformation of basic form intohydrochloride, the product was dissolved in CH₂Cl₂-methanol mixture.(The mixture was added until the product was completely dissolved.)Then, a solution of hydrochloric acid in dioxane was added. The solutionwas evaporated to dryness. A dark-lilac precipitate obtained was washedwith methanol and acetonitrile. Yield of Example 6 as hydrochloride:0.118 g.

H¹-NMR (400 MHz, DMSO-d₆) spectrum: δ 0.88 (3H, t, J=7.32 Hz); 1.53-1.62(2H, m); 2.13-2.20 (2H, m); 2.73 (2H, s); 2.80-2.83 (4H, m); 2.95-2.96(2H, m); 3.65-3.68 (2H, m); 4.68 (2H, t, J=7.3 Hz); 7.81 (1H, d, J=8.6Hz); 7.85 (1H, d, J=8.6 Hz); 8.15 (1H, dd, J=8.6 Hz, J=1.3 Hz); 8.70(1H, br. s); 8.71 (1H, d, J=1.3 Hz). ELSD: 100%, ESI-MS: m/z 362 [M+H]⁺.

Example 7

N-(2-Hydroxyethyl)-2-nitrobenzenesulfonamide (54)

Ethanolamine (10 mL, 165 mmol) was dissolved in ethyl acetate (60 mL).Then a solution of Na₂CO₃ (22.7 g, 214.2 mmol) in water (100 mL) wasadded. Then, a solution of 2-nitrobenzenesulfonyl chloride (35.0 g,157.9 mmol) in ethyl acetate (100 mL) was added dropwise under stirring.The resulting solution was stirred at room temperature for 2 h (TLCmonitoring, eluent: CH₂Cl₂). The organic layer was separated, washedwith water and a solution of citric acid, dried over Na₂SO₄, andevaporated. Yield of 54: 29.5 g (73%) as white crystals.

2-{[(2-Nitrophenyl)sulfonyl]amino}ethyl methanesulfonate (55)

Triethylamine (20 mL, 144.6 mmol) was added to a solution of compound 54(29.5 g, 119.9 mmol) in ethyl acetate (300 mL). The mixture was cooledto 10° C. in an ice bath. Then, a solution of mesyl chloride (10.2 mL,131.8 mmol) in ethyl acetate (50 mL) was added dropwise at a temperature≤20° C. The resulting solution was stirred at room temperature for 3 h(TLC monitoring, eluent: hexane-ethyl acetate, 4:1). The reactionmixture was filtered to remove the precipitate—triethylaminehydrochloride. The filtrate was washed with aqueous NaHCO₃, water, andevaporated. Yield of 55:32.2 g (83%) as crystals.

1-[(2-Nitrophenyl)sulfonyl]aziridine (56)

A solution of KOH (1.73 g, 31 mmol) in water (50 mL) was added to asolution of compound 55 (10 g, 31 mmol) in ethyl acetate (100 mL). Theaqueous layer stained in yellow. The mixture was stirred at roomtemperature for 1 h (TLC monitoring, eluent: CH₂Cl₂-ethyl acetate, 1:1).If necessary, additional portions of KOH solution (0.5 eq.) were added.The organic layer was separated, washed thoroughly with water, asolution of citric acid (to pH 7), again with water, dried over Na₂SO₄,and evaporated. Yield of 56: 6.6 g (93%) as a yellowish fluid oil.

N-[2-(9H-Carbazol-9-yl)ethyl]-2-nitrobenzenesulfonamide (51)

Carbazole (1.10 g, 6.59 mmol) was dissolved in CH₃CN (40 mL). Thensodium hydrate (0.33 g, 8.25 mmol) was added, and the mixture wasstirred at room temperature for 15-20 min. Then, a solution of aziridine56 (1.5 g, 7.89 mmol) in CH₃CN (30 mL) was added in one portion. Theresulting mixture was stirred for 1 h (TLC monitoring, eluent:hexane-ethyl acetate, 1:1), poured into water, acidified with HCl to pH1, and stirred again at room temperature. Gradually, an orange productprecipitated from the turbid solution. The precipitate was filtered, andwashed with MeOH and ether. Yield of 51: 2.15 g (83%) as orangecrystals.

N-[2-(3,6-Diacetyl-9H-carbazol-9-yl)ethyl]-2-nitrobenzenesulfonamide(52)

Compound 51 (5.0 g, 12.7 mmol) was dissolved in PhNO₂ (30 mL). Thesolution was cooled in an ice bath. Then AlCl₃ (8.5 g, 63.7 mmol) andafter that AcCl (4.5 mL, 63.1 mmol) were added. The mixture was kept for40 min (LC/MS monitoring), diluted with water, extracted withchloroform, and evaporated. The product was purified on a short thickcolumn with silica gel, eluent: CH₂Cl₂-ethyl acetate 100:0→50:50. Amixture of ethanol and 25% aqueous ammonia (4:1) was added to theresidue. The resulting product was refluxed for 1.5-2 h. A hotsuspension was filtered off. Product 52 was isolated (4.11 g, 68%) asbeige crystals.

1,1′-[9-(2-Aminoethyl)-9H-carbazole-3,6-diyl]diethanone (53)

Compound 52 (4.11 g, 8.58 mmol) was dissolved under reflux in a mixtureof CH₃CN (150 mL) and methanol (50 mL). Then, Cs₂CO₃ (8.4 g, 25.78 mmol)was added and at once PhSH (2.6 mL, 25.48 mmol) was poured in. Theresulting mixture was refluxed for 2.5 h (TLC monitoring, eluent:chloroform-methanol, 9:1) and evaporated to dryness. Then, water and asolution of HCl were added. As a result, all the solid products weredissolved. The acidic aqueous solution was extracted with ethyl acetateuntil the latter ceased to become yellow. The aqueous layer wasneutralized with a saturated solution of NaHCO₃, extracted with aCH₂Cl₂-MeOH mixture (4:1), and the extract was evaporated. The residuewas washed off with MeCN and washed with ether. Yield of 53: 1.25 g(50%) as beige crystals.

1,1′-{9-[2-(Isopropylamino)ethyl]-9H-carbazole-3,6-diyl}diethanone(Example 7; 6h)

Acetone (5 mL, 68.10 mmol) was added to a solution of compound 53 (1.25g, 4.25 mmol) in CH₂Cl₂ (50 mL). Then STAB (3.0 g, 14.15 mmol) wasadded. The mixture obtained was stirred at room temperature for 4.5 h(TLC monitoring, eluent: CHCl₃-MeOH, 9:1) and then poured into anaqueous solution of NaHCO₃. The organic layer was separated. The aqueouslayer was extracted with CH₂Cl₂. The product was dried over Na₂SO₄ andevaporated. The residue was purified on a column, eluent: CHCl₃-MeOH99:1→90:10. Example 7 (1.04 g, 73%) was isolated as a rapidlycrystallizing oil. For the preparation of its hydrochloride, the basewas dissolved in CH₂Cl₂. Then, a solution of HCl in dioxane was added.The mixture was evaporated to dryness. The product was washed withether.

¹H NMR (DMSO-d₆) spectrum: δ 1.25 (6H, d, J=6.6 Hz); 2.72 (6H, s);3.33-3.39 (3H, m); 4.87 (2H, t, J=7.3 Hz); 7.92 (2H, d, J=8.7 Hz); 8.17(2H, dd, J=8.7 Hz, J=1.6 Hz); 9.01 (2H, d, J=1.6 Hz); 9.24 (1H, br. s);9.33 (1H, br. s). ELSD: 100%, ESI-MS: m/z 336 [M+H].⁺

Example 8

The synthesis of compound 51 was described in Example 7.

N-{2-[3,6-bis(3-chloropropanoyl)-9H-carbazol-9-yl]ethyl}-2-nitrobenzenesulfonamide(57)

Compound 51 (20.0 g, 50.6 mmol) was dissolved in PhNO₂ (130 mL) and thesolution was cooled in an ice bath. Then AlCl₃ (34.0 g, 254.7 mmol) andafter that, 3-chloropropionyl chloride (25.0 mL, 259.8 mmol) were added.The mixture was kept for 40 min (LC/MS monitoring), poured in a mixtureof diluted HCl and ice, extracted with CHCl₃, and left for 16 h. Thestraw-colored precipitated obtained was filtered and washed with ether.Yield of 57: 18.47 g (63%).

N-[2-(3,9-dioxo-1,2,3,9,10,11-hexahydro-6H-dicyclopenta[c,g]carbazol-6-yl)ethyl]-2-nitrobenzenesulfonamide(58)

Sulfuric acid (200 mL) was heated to 40° C. Then, compound 57 (18.47 g,32.12 mmol) was added in portions. The mixture was heated to 90° C. andkept at this temperature for 1.5-2 h (TLC monitoring, eluent:CH₂Cl₂-ethyl acetate, 4:1). The resulting mixture was poured onto ice.The grey precipitate was filtered off, washed on a filter with a mixtureof CHCl₃-MeOH (4:1). The remaining precipitate (on a filter) wasrecrystallized with DMF, washed with CH₃CN and ether. Yield of pure 58:2.2 g (14%).

6-(2-aminoethyl)-10,11-dihydro-1H-dicyclopenta[c,g]carbazole-3,9(2H,6H)-dione(59)

Compound 58 (2.2 g, 4.38 mmol) was suspended in a mixture of chloroform(200 mL) and methanol (200 mL). Then Cs₂CO₃ (4.3 g, 13.20 mmol) and atonce PhSH (1.3 mL, 12.74 mmol) were added. The solution was refluxed for18 h (LC/MS monitoring) and evaporated to dryness. Then, an aqueoussolution of citric acid was added. The solution obtained was neutralizedwith an aqueous NaHCO₃ solution. The precipitate was filtered off,washed with CH₃CN and ether. The product isolated was used in the nextstep without purification.

6-[2-(isopropylamino)ethyl]-10,11-dihydro-1H-dicyclopenta[c,g]carbazole-3,9(2H,6H)-dione(Example 8)

Crude compound 59 (1.69 g) was suspended in CH₂Cl₂ (200 mL). Thenacetone (4 mL) and STAB (3.4 g) were added. The mixture was stirred for24 h (TLC monitoring, eluent: CHCl₃-MeOH, 9:1), then poured into anaqueous NaHCO₃ solution. Then, another portion of CH₂Cl₂ and MeOH wasadded. The organic layer was separated, evaporated, washed with MeCN andether. Yield of Example 8: 0.759 g. For the preparation ofhydrochloride, the isolated product was suspended in a mixture of CH₂Cl₂and MeOH, then a solution of HCl in dioxane was added. The resultingmixture was evaporated to dryness, and ethanol was added to the residue.The ethanolic solution was refluxed and cooled, and the precipitate wasfiltered. Yield of compound Example 8 hydrochloride: 0.684 g. ¹H-NMR(DMSO-D₆) spectrum: δ 1.24 (6H, d, J=6.6 Hz); 2.77-2.79 (4H, m);3.34-3.43 (3H, m); 3.81-3.84 (4H, m); 4.91 (2H, t, J=7.3 Hz); 7.83 (2H,d, J=8.6 Hz); 7.91 (2H, d, J=8.6 Hz); 9.11 (2H, br. s). ELSD: 100%,ESI-MS: m/z 360 [M+H].⁺

Example 9

N-Ethyl-2-nitrobenzenesulfonamide (63)

A solution of 2-nitrobenzenesulfonyl chloride (140 g, 361 mmol) in ethylacetate (250 mL) was added dropwise at room temperature to a mixture of70% aqueous ethylamine (50 mL, 630 mmol), ethyl acetate (100 mL), Na₂CO₃(67 g, 632 mmol), and water (250 mL). The reaction mixture was stirredfor 4 h (TLC monitoring, CH₂Cl₂). The organic layer was separated,washed with water, with a solution of citric acid, dried with Na₂SO₄,and evaporated. The residue solidified into a white crystalline mass.The latter was triturated with hexane, filtered off, and dried to give134 g (92%) of the product.

N-(3-Bromopropyl)-N-ethyl-2-nitrobenzenesulfonamide (64)

Compound 63 (7.8 g, 33.9 mmol) was dissolved in DMF (100 mL).1,3-Dibromopropane (35 mL, 343.0 mmol) and then in portions NaH (2.7 g,67.5 mmol) were added, whereupon the temperature rose to 50-60° C. Thereaction mixture was stirred at this temperature for 1 h (TLCmonitoring, CH₂Cl₂), carefully poured into ice/water mixture, andextracted with ethyl acetate. The extract was evaporated. The residuewashed with hexane to remove the 1,3-dibromopropane. Compound 64 (9.7 g,82%) was obtained as a viscous yellow oil.

N-[3-(9H-Carbazol-9-yl)propyl]-N-ethyl-2-nitrobenzenesulfonamide (60)

Carbazole (4.15 g, 24.8 mmol) and bromide 64 (9.7 g, 27.6 mmol) weredissolved in DMF (30 mL). NaH (2.0 g, 50.0 mmol, 2 eq) was added inportions, whereupon the temperature rose to 60° C. The reaction mixturewas stirred for 1 h (TLC monitoring, hexane/ethyl acetate 3:2), pouredinto ice/water mixture, and extracted with ethyl acetate. The residuewas triturated with ether. The yellow precipitate was filtered off anddried to afford 6.65 g (61%) of the product.

N-{3-[3,6-Bis(3-chloropropanoyl)-9H-carbazol-9-yl]propyl}-N-ethyl-2-nitrobenzenesulfonamide(61)

Compound 63 (6.65 g, 15.2 mmol) was dissolved in PhNO₂ (70 mL). Thesolution was cooled in an ice bath. AlCl₃ (12.2 g, 91.4 mmol) and then2-chloropropionyl chloride (8.8 mL, 91.4 mmol) were added. The reactionmixture was kept for 40 min (LC/MS monitoring), diluted with water, andextracted with CH₂Cl₂. The extract was evaporated. The residue waspurified by chromatography in a short thick column (eluent: CH₂Cl₂/ethylacetate 0:100→50:50. The residue was triturated with ether, filteredoff, and dried to give 7.60 g (81%) of a greenish crystalline product.

N-[3-(3,9-Dioxo-1,2,3,9,10,11-hexahydro-6H-dicyclopenta[c,g]carbazol-6-yl)propyl]-N-ethyl-2-nitrobenzenesulfonamide(62)

H₂SO₄ (110 mL) was heated to 40° C. Compound 61 (7.6 g, 12.3 mmol) wasadded in portions. The reaction mixture was heated to 100° C., kept atthis temperature for 2 h (TLC monitoring, CH₂Cl₂/ethyl acetate 4:1), andpoured into ice. The formed gray precipitate was filtered off. ThenCHCl₃/MeOH 4:1 (500 mL) was poured into the filter. The solid on thefilter dissolved, and the formed solution was transferred into aseparation funnel. An aqueous solution of Na₂CO₃ was added. The organiclayer was separated, dried with Na₂SO₄, and evaporated. Acetonitrile wasadded to the residue. A beige precipitate was filtered off, washed withacetonitrile, with ether, and dried to give 1.56 g (23%) of a beigecrystalline product.

6-[3-(Ethylamino)propyl]-10,11-dihydro-1H-dicyclopenta[c,g]carbazole-3,9(2H,6H)-dione(Example 9)

Compound 62 (1.56 g, 2.86 mmol) was suspended in a mixture of CHCl₃ (80mL) and MeOH (80 mL). Cs₂CO₃ (2.8 g, 8.59 mmol) and then immediatelyPhSH (0.87 mL, 8.53 mmol) were added. The reaction mixture was refluxedfor 6 h (TLC monitoring, CHCl₃/MeOH 9:1) and evaporated to dryness.Aqueous citric acid was added. The solution was neutralized with asolution of NaHCO₃. The formed precipitate was filtered off, washed withethyl acetate, acetonitrile, water, again with acetonitrile, and withether to give 0.75 g (73%) of the product. The latter was transformed toits hydrochloride. After evaporation with 4M HCl in dioxane the residuewas washed with MeOH to afford 0.59 g of the hydrochloride. ¹H NMR(DMSO-d₆): δ 1.66 (3H, t, J=7.3 Hz); 2.09-2.17 (2H, m); 2.73-2.77 (4H,m); 2.86-2.91 (2H, m); 2.95-3.10 (2H, m); 3.77-3.80 (4H, m); 4.70 (2H,t, J=7.3 Hz); 7.80 (2H, d, J=8.6 Hz); 7.89 (2H, d, J=8.6 Hz); 8.83 (2H,br.s). ELSD: 100%, ESI-MS: m/z 360 [M+H]⁺.

Examples 10 and 11

The synthesis of compound 46 is described in Example 6

6-[2-(1-methylpyrrolidin-2-yl)ethyl]-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(65)

Carbazole 46 (0.4 g, 1.81 mmol) was dissolved in CH₂Cl₂ (7 mL), then NaH(0.22 g, 5.50 mmol) was added. The mixture was stirred at roomtemperature for 5-10 min, and 2-(2-chloroethyl)-1-methylpyrrolidinehydrochloride (0.4 g, 2.17 mmol) was added. The mixture was heated to60° C. and kept at this temperature for 4 h (TLC monitoring, eluent:CHCl₃-MeOH, 9:1). The resulting mixture was poured into water. Theproduct was extracted with ethyl acetate, dried over Na₂SO₄, andevaporated. The residue was triturated with ether. The precipitate wasfiltered off. Yield of 65: 0.365 g (61%)—S-isomer and 0.336 g(55%)—R-isomer.

9-acetyl-6-[2-(1-methylpyrrolidin-2-yl)ethyl]-1,6-dihydrocyclopenta[c]carbazol-3(2H)-one(Examples 10 and 11)

Compound 65 (0.336 g, 1.00 mmol) was dissolved in PhNO₂ (7 mL). Thesolution was cooled down in an ice bath. Then, AlCl₃ (0.67 g, 5.02 mmol)and after that, AcCl (0.36 mL, 5.04 mmol) were added. The resultingmixture was kept for 40 min (LC/MS monitoring), diluted with water,neutralized with aqueous NaHCO₃ solution, extracted with CHCl₃, andevaporated. The product was purified on a short thick column, eluent:100:0→90:10. Yield of product: 0.307 g (82%) (R); S-isomer was obtainedsimilarly (77%).

Example 10

¹H-NMR (DMSO-D₆) spectrum: δ 1.69-1.79 (1H, m); 1.86-2.01 (2H, m);2.07-2.24 (2H, m); 2.42-2.56 (1H, m); 2.73 (3H, s); 2.75 (3H, d, J=5.12Hz); 2.79-2.82 (2H, m); 2.96-3.05 (1H, m); 3.36-3.43 (1H, m); 3.49-3.57(1H, m); 3.64-3.67 (2H, m); 4.62-4.75 (2H, m); 7.80 (1H, d, J=8.6 Hz);7.87 (1H, d, J=8.6 Hz); 7.96 (1H, d, J=8.6 Hz); 8.19 (1H, dd, J=8.6 Hz,J=1.5 Hz); 8.70 (1H, d, J=1.5 Hz); 10.63 (1H, br. s). ELSD: 100%,ESI-MS: m/z 374 [M+H]⁺.

Example 11

¹H-NMR (DMSO-D₆): δ 1.69-1.79 (1H, m); 1.86-2.01 (2H, m); 2.05-2.24 (2H,m); 2.40-2.46 (1H, m); 2.73 (3H, s); 2.75 (3H, d, J=5.12 Hz); 2.79-2.82(2H, m); 2.98-3.02 (1H, m); 3.36-3.43 (1H, m); 3.47-3.57 (1H, m);3.63-3.66 (2H, m); 4.62-4.75 (2H, m); 7.80 (1H, d, J=8.6 Hz); 7.87 (1H,d, J=8.6 Hz); 7.96 (1H, d, J=8.8 Hz); 8.18 (1H, dd, J=8.8 Hz, J=1.4 Hz);8.69 (1H, d, J=1.4 Hz); 10.72 (1H, br. s). ELSD: 100%, ESI-MS: m/z 374[M+H]⁺.

Synthesis of Example 50

Step 1. 4-hydroxyindan-1-one (86)

Chromanone 85 (100 g, 0.68 mol) was added dropwise to mixture 150 g NaCland 500 g AlCl₃ at 150° C. The resulting mixture was heated to 180° C.,then stirred for 8 h. The mixture then was cooled to room temperatureand 1 kg of crushed ice was added with intensive stirring. Afterhomogenization, the mixture was filtered, washed with IL cold water, anddried to give about 80 g (80%) 4-hydroxyindan-1-one (86) as gray solid.

Step 2. 1-oxo-2,3-dihydro-1H-inden-4-yl trifluoromethanesulfonate (87)

Triflic anhydride (152 g, 0.54 mol) was added dropwise to solution 80 gof compound 86 and 60 g of NEt₃ in 1 L CH₂Cl₂ at 0° C. The resultingmixture was refluxed for 2 h, cooled to room temperature, filtered,washed with water (0.5 L), aqueous citric acid (50 g/0.5 L), brine (0.5L), dried on Na₂SO₄, and evaporated to give about 100 g (67%) ofcompound 87 as dark liquid.

Step 3. 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indan-1-one (88)

A mixture consisting of potassium acetate (48 g, 0.5 mol, 1.5 eq),triflate 87 (100 g, 0.36 mol, 1.1 eq), bis(pinacolo)diborane (100 g,0.38 mol, 1.2 eq), PPh₃ (5 g, 0.02 mol, 0.06 eq), and PdCl₂(PPh₃)₂ (6.9g, 0.01 mol, 0.03 eq) in degassed toluene (2 L) was refluxed under argonatmosphere overnight. The resulting mixture was evaporated to drynessand chromatographed with eluted by EtOAc/hexanes 1/5. Fractionscontaining desired product was collected and evaporated to give about 50g (57%) of compound 88 as light-yellow solid.

Step 4. 4-(2-nitrophenyl)-indan-1-one (89)

Degassed toluene (0.5 L), borolan 88 (10 g, 39 mmol),2-chloro-1-nitrobenzene (6.3 g, 40 mmol), Pd(PPh₃)₄ (3 g, 2.6 mmol) and2M Na₂CO₃ (200 mL) were mixed under a rapid flow of argon, and theresulting mixture was refluxed overnight. Water (200 mL) and EtOAc (500mL) were added. The aqueous layer was separated and extracted with EtOAc(2×500 mL), and the combined extracts dried over Na₂SO₄. The solvent wasevaporated at reduced pressure and the crude product purified by columnchromatography (20% EtOAc in hexanes) to give 9 g (91%) of the biphenylintermediate 89 as a yellow solid.

Step 5. 1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (90)

Biphenyl 89 (9 g, 35 mmol) and triethylphosphite were added to a sealedbomb and heated at 120° C. overnight. After cooling to room temperature,the mixture was filtered to give 4 g of1,2-dihydro-6H-cyclopenta[c]carbazole-3-one. Precipitation with diethylether yielded an additional 2 g of desired product. Common yield 90 asyellow powder was 77.5%.

Step 6. 9-acetyl-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (91)

1,2-dihydro-6H-cyclopenta[c]carbazole-3-one 90 (6 g, 27.1 mmol) wassuspended in dry CH₂Cl₂ (100 mL). Anhydrous AlCl₃ (6 g, 45 mmol) wasadded with stirring on ice bath, followed by the dropwise addition ofAcCl (2.4 mL, 33.2 mmol). The reaction mixture was stirred at about 5°C. for 24 h, and poured into ice water. The precipitated grey solid wasfiltered off, washed with water (2×100 mL), acetone (3×50 mL), diethylether (3×50 mL) to give 4 g (56.3%) of9-acetyl-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one 91.

Step 7.9-acetyl-1,2-dihydro-6-(2-bromoethyl)-cyclopenta[c]carbazole-3-one (92)

NaH (2 g of 60%, 50 mmol) was added to a suspension of compound 91 (4 g,15.2 mmol) in dry DMF (100 mL), and the mixture was stirred at roomtemperature about 1 h until the evolution of hydrogen ceased.1,2-dibromoethane (20 g, 106 mmol) was added in dropwise under nitrogen.The reaction mixture was stirred for 30 min at room temperature, then at60° C. for 24 h, and poured into ice water (300 mL). The precipitatedgrey solid was filtered off and the crude product purified by columnchromatography (CHCl₃) to give 2 g of starting material and about 1 g(2.7 mmol, 35.5% from converted material) of the alkylated carbazole 92as a cream solid.

Step 8. 9-acetyl-1,2-dihydro-6-(2-(2-propyl)aminoethyla)-cyclopenta[c]carbazole-3-one hydrochloride (Example 50)

Bromide 92 (1 g, 2.7 mmol) and 2-propylamine were added to sealed bomband heated at 180° C. overnight. After cooling to room temperature, themixture was evaporated to dryness, the residue was worked up with 100 mlsaturated water solution of sodium bicarbonate and filtered. Filter cakewas purified by column chromatography (CHCl₃/MeOH 10/1). Fractionscontaining desired product were collected, evaporated to dryness,diluted with 40% water HCl (10 mL), evaporated to dryness andreevaporated with toluene (2×100 mL) to give 200 mg (0.52 mmol, 19%) of9-acetyl-1,2-dihydro-6-(2-(2-propyl)aminoethyl)-cyclopenta[c]carbazole-3-one hydrochloride (Example 50) as a gray solid.

1H-NMR (DMSO-d₆): 1.23 d (6H), 2.74 s (3H), 2.83 m (2H), 3.67 m (2H),4.87 m (2H), 7.85 dd (2H), 7.97 d (1H), 8.22 d (1H), 8.71 s (1H),8.9-9.2 wide s (2H).

Synthesis of Example 51

Step 1. 9-(3-dimethylaminopropyl)-carbazole (2b)

NaH (14.4 g of 60%, 360 mmol) was added portion-wise under argon to asuspension of carbazole 1 (20 g, 119.8 mmol) in dry DMF (200 mL). Themixture was stirred at room temperature for about 1 h until theevolution of the hydrogen ceased. 3-Dimethylaminopropylchloridehydrochloride (28 g, 180 mmol) was added portion-wise at roomtemperature. The reaction mixture was stirred for 20 min at roomtemperature, then at 60° C. for 3 hrs, and poured into cold water (500mL). The product was extracted with EtOAc (2×500 mL). The organic layerwas back extracted with 10% HCl (200 mL) and the acidic layer wasextracted with EtOAc to remove the unreacted carbazole. Saturatedammonia (200 mL) was added and the product extracted again with EtOAc.Evaporation of the solvent afforded 25 g (99 mmol, 83%) of9-(2-dimethylaminopropyl)carbazole 2b as a brown oil.

Step 2.3,6-di(4-chloropropan-2-one-yl)-9-(2-dimethylaminopropyl)carbazole (93)

9-(2-Dimethylaminopropyl)carbazole 2b (25 g, 99 mmol) was dissolved indry CH₂Cl₂ (150 mL). Anhydrous AlCl₃ (40 g, 300 mmol) was addedportion-wise with stirring and cooling in an ice bath.3-Chloro-propionyl chloride (30 mL, 312 mmol) was added dropwise, whilemaintaining the internal temperature below 5° C. The reaction mixturewas stirred at this temperature overnight, poured into crushed ice andextracted with CHCl₃/MeOH 1/1 (3×500 mL). The extracts were evaporatedto dryness, and the resulting green oil was triturated with diethylether (2×250 mL) to give 20 g (46 mmol, 47%) of3,6-di(3-chloropropionyl)-9-(2-dimethylaminopropyl)-carbazole 93 as graypowder.

Step 3.6-(3-Dimethylaminopropyl)-1,2,10,11-tetrahydro-6H-bis-(cyclopenta[c]carbazol-3,9-dione(Example 51)

3,6-Di-(3-chloropropionyl)-9-(2-dimethylaminopropyl)carbazole 93 (20 g,46 mmol) was added portion-wise to trifluoromethanesulfonic acid (100 g)with stirring at room temperature and the reaction mixture was heated to95° C. overnight. After cooling to room temperature, the reactionmixture was poured into ice water. The precipitate was filtered off,worked up with saturated sodium bicarbonate, and filtered off again. Theresulting filter cake contained about 80% desired product and 20%isomer. After a single crystallization from methanol, the precipitatewas worked up with 4N HCl/dioxane (100 ml), evaporated to dryness andrecrystallized from EtOH to give about 2.8 g (7.1 mmol, 15.4%) of 95%Example 51 as white solid.

LCMS: 100%; 1H-NMR (DMSO-d₆) 95%: 2.18 m (2H), 2.71 s (6H), 2.78 m (4H),3.15 m (2H), 3.83 m (4H), 4.68 m (2H), 7.85 dd (4H), 10.0 br s (1H).

Alternative Synthesis of Examples 15 and 17 and Synthesis of Example 20

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Proton nuclear magnetic resonance spectra wereobtained on a Bruker ARX 400 spectrometer at 400 MHz. The solvent peakwas used as the reference peak for proton spectra. The progress ofreactions was controlled by TLC or/and LC-MS analysis.

9-(2-Diethylaminoethyl)-carbazole (68)

NaH (14.4 g of 60%, 360 mmol, 3 eq) was added portion-wise under argonto a suspension of carbazole (1) (20 g, 119.8 mmol, 1 eq) in dry DMF(200 mL). The mixture was stirred at room temperature for 30 min untilthe evolution of the hydrogen ceased. 2-Diethylaminoethylchloridehydrochloride (31 g, 180 mmol, 1.5 eq) was added in portion-wise at roomtemperature. The reaction mixture was stirred for 20 min at roomtemperature, then at 60° C. for 3 hrs (TLC monitoring), and poured intocold water (1 L). The product was extracted with EtOAc (5×200 mL); theorganic layer was back extracted with 10% HCl (400 mL) and acidic layerwas extracted with EtOAc to remove the un-reacted carbazole. The pH ofaqueous layer was adjusted to about 9 with K₂CO₃ and the productextracted again with EtOAc. Evaporation of the solvent afforded 29.5 g(93%) of 9-(2-diethylaminoethyl)carbazole (68) as a brown oil.

3,6-Di(4-chloropropan-2-one-yl)-9-(2-diethylaminoethyl)carbazolehydrochloride (69)

9-(2-Diethylaminoethyl)carbazole (68) (17.9 g, 67.3 mmol, 1 eq) wasdissolved in dry dichloromethane (150 mL). Anhydrous AlCl₃ (45 g, 337mmol, 5 eq) was added portion-wise with stirring and cooling in an icebath. 3-chloro-propionyl chloride (32.4 mL, 337 mmol, 5 eq) was addeddropwise, while maintaining the internal temperature below 5° C. Thereaction mixture was stirred at this temperature for 15 h, poured intocold 3% HCl (violent foaming should be avoided) and extracted with CHCl₃(5×500 mL). The extracts were evaporated and the resulted green oiltriturated with diethyl ether (5×50 mL) to give 24 g (74%) of3,6-di(3-chloropropionyl)-9-(2-diethylaminoethyl)-carbazolehydrochloride (69) as dark grey solid.

¹H-NMR (DMSO-d₆): 1.27 (t, 6H), 2.72 (q, 4H), 3.75 (m, 6H), 4.02 (m,4H), 5.00 (t, 2H), 8.00 (d, 2H), 8.17 (d, 2H), 9.16 (s. 2H), 11.42 (s,1H).

6-(3-Diethylaminoethyl)-1,2,10,11-tetrahydro-6H-bis-(cyclopenta[c]carbazol-3,9-dione(70)

3,6-Di-(3-chloropropionyl)-9-(2-diethylaminoethyl)carbazolehydrochloride (69) (5 g, 10.33 mmol, 1 eq) was added portion-wise totrifluoromethanesulfonic acid (50 g, 333 mmol, 32 eq) with stirring atroom temperature and the reaction mixture was heated to 95° C.overnight. After cooling to room temperature, the reaction mixture waspoured into ice water. The pH of the aqueous solution was adjusted to 9with 10% NaOH and the product extracted with EtOAc/THF=3/1 mixture. Thesolvents were evaporated out and the crude product purified by columnchromatography (10% EtOH in EtOAc) to give 1.12 g (29%) of pureintermediate 70 as white solid.

MS(ESI): m/z=375.3 [M+H]⁺; ¹H-NMR (DMSO-d₆): 0.70 (t, 6H), 2.41 (q, 4H),2.68 (m, 6H), 3.73 (m, 4H), 4.55 (t, 2H), 7.77 (s, 4H).

Example 15

Intermediate 70 (1.12 g, 2.99 mmol, 1 eq) was dissolved in CH₂Cl₂ (20mL) and 10% HCl solution in ethanol (3.4 g, 93.15 mmol, 30 eq) was addedcausing formation of a voluminous precipitate. The solvents wereevaporated at reduced pressure and the residue triturated with hot MeOHto give 1.17 g (98%) of Example 15 as an off-white solid.

Purity: 97.3% by HPLC; MS(ESI): m/z=375.3 [M−HCl+H]⁺; m.p.=312.1-314.7°C.

Examples 17 and 20 are prepared by an identical process using the properchloroamine, i.e., (CH₃)₂CH—NH—H₂CH₂—Cl or (C₂H₅)NHCH₂CH₂—Cl.

Alternative Synthesis of Example 7 (Compound 6h)

Experimental

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Proton nuclear magnetic resonance spectra wereobtained on a Bruker ARX 400 spectrometer at 400 MHz. The solvent peakwas used as a reference peak for proton spectra. TLCs were run onsilica, unless otherwise noted.

3,6-Diacetylcarbazole (4)

Carbazole (20 g, 0.12 mol, 1 eq) was suspended in anhydrous CH₂Cl₂ (300mL) under argon and the resulting mixture cooled to 0° C. AlCl₃ (95.5 g,0.72 mol, 6 eq) was added to the mixture followed by the drop-wiseaddition of acetyl chloride (25.5 mL, 0.36 mol, 3 eq), while maintainingthe internal temperature at about 0° C. After 3 hours stirring at 0° C.,another portion of acetyl chloride (5 mL, 0.07 mol, 0.6 eq) was addedand the stirring continued for another 2 hours.

The reaction mixture was poured into ice while stirring. The solid wascollected by filtration, washed with CH₂Cl₂ then water and dried in avacuum oven at 50° C. Yield 16.3 g (54%) of carbazole 4. The filtrateand washings were combined and extracted with CH₂Cl₂ (3×300 mL). TheCH₂Cl₂ extracts were washed with saturated NaHCO₃ (1×300 mL) and brine(1×300 mL). After drying over Na₂SO₄, the filtrate was evaporated todryness to give the crude product, which was used without furtherpurification.

MS(ESI): m/z=252.0 [M+H]⁺

2-N-Boc-2-N-isopropylaminoethanol (14)

(Boc)₂O (50.8 g, 0.23 mol, 2 eq) was added to a solution of2-N-isopropylaminoethanol (72) (22.3 mL, 70%, 0.136 mol) in MeOH (200mL). The reaction mixture was stirred at ambient temperature for 4 hoursthen diluted with water (1.2 L) and the product extracted with EtOAc(4×400 mL). The combined EtOAc extracts were washed with brine (1×400mL) and dried over Na₂SO₄. The solvent was evaporated out and the crudeproduct purified by column (eluent Hexanes: EtOAc from 4:1 to 1:1) togive 19.5 g (70.5% yield) of pure 73 as viscous oil.

3-Isopropyl-2-oxazolidinone (74)

MsCl (8.5 mL, 0.109 mol) was added dropwise to a solution of the alcohol(73) (18.5 g, 0.091 mol) and TEA (19 mL, 0.137 mol) in anhydrous THF(200 mL) cooled to −20° C. with vigorous stirring. The reaction wasallowed to warm up slowly to ambient temperature (3 hours). The solidswere removed by filtration and washed with THF (20 mL). Saturated Na₂CO₃(100 mL) was added to the filtrate and the resulting mixture stirred atambient temperature overnight, then diluted with water (200 mL) andextracted with EtOAc (3×200 mL). The combined EtOAc extracts were washedwith 1% HCl (1×200 mL), brine (1×200 mL) an dried over Na₂SO₄. Thesolvent was evaporated out to give 8.8 g (75% yield) of cyclic carbamate(74), which was used further without purification.

MS(ESI): m/z=130.1 [M+H]⁺

3,6-Diacetyl-9-(2-N-isopropylaminoethyl)-carbazole (71)

A mixture of 3,6-diacetylcarbazole (4) (15.12 g, 0.06 mol),3-isopropyl-2-oxazolidinone (74) (7.80 g, 0.06 mol), Cs₂CO₃ (39.2 g,0.12 mol), and DBU (9 mL, 0.06 mol) in NMP (150 mL) was stirred at 190°C. for 24 hours, then poured into H₂O (500 mL) and extracted with EtOAc(3×300 mL). The combined EtOAc extracts were washed with brine (2×200mL) and dried over Na₂SO₄. The solvent was evaporated and the crudeproduct purified by column chromatography (eluent—(5 to 20%) MeOH(containing 0.1% of NH₃H₂O)/EtOAc). The first fractions contained theun-reacted starting material (˜1.7 g). The pure fractions were combinedand solvent evaporated to dryness to give 9.36 g (contained 20% EtOAc byNMR) of pure (71). The less pure fractions were combined, evaporated todryness and the residue triturated with EtOAc to give another 3.47 g ofthe pure product. Total yield—54.5% (after subtracting the amount ofEtOAc); (61.6% yield based on reacted starting material).

MS(ESI): m/z=337.1 [M+H]⁺

Compound 6h

Intermediate (71) (3.47 g, 0.0103 mol) was dissolved in CH₂Cl₂ (30 mL)and the resulting solution cooled to about 5° C. 10% HCl/EtOH solution(5.6 g, 0.0153 mol) was added dropwise with stirring. After 40 minutesthe solvent was removed under vacuum, the residue was dried under highvacuum at 40° C. to give 3.5 g (93% yield) of 6h. Similarly, the othercrop of 71 (9.36 g, 20% EtOAc) was converted into 6h.

Analysis Data: Purity: 99.5% by HPLC; MS(ESI): m/z=337.3 [M−HCl+H]⁺;m.p.=292.7-294.1° C.

Alternative Synthesis of Example 4

Step 1.

4-Hydroxy-1-indanone (76) was synthesized by aluminum chloride inducedre-cyclization of dihydrocoumarin (75), according to Org. Lett., 2007,9(15), p. 2915-2918. The reaction was performed on 100 g of (75) tofurnish (76) with 85% yield.

Step 2.

The indanone (76) was converted to triflate (77) with 90% yield byreaction with triflic anhydride in CH₂Cl₂ in the presence of Py,followed by flash chromatography.

Step 3.

Triflate (79) was synthesized in the same manner with quantitativeyield, starting from 50 g of 4-methoxy-2-nitrophenol (78).

Steps 4 and 5.

Following the protocol for a one-pot synthesis of biphenyl compoundsfrom Synthetic Communications, 2006, 36 p. 3809-3820, intermediate (81)was obtained in 80% yield starting from 60 g of triflate (77).

Step 6.

The cyclization of the biphenyl intermediate (81) by heating with excessof triphenylphosphine in 1,2-dichlorobenzene was carried out on a smallscale first to afford the expected carbazole (82) with ˜50% yield(un-optimized) after precipitation from the reaction mixture with ether.The same reaction was scaled up to 42 g of (81), yielding 27 g (73%yield) of pure (82).

Step 7.

The acetylation of the intermediate (82) (27.8 g scale) was carried outin CH₂Cl₂ according to the standard protocol to give 26 g (80% yield) ofthe intermediate (83).

Step 8.

After an exhaustive extraction with a 1:1 mixture of EtOAc/THF, about68% yield of the crude product (84), containing a baseline impurity byTLC, was obtained. It was used for the de-methylation step withoutfurther purification.

Step 9.

The removal of the methyl group from (84) was accomplished by heatingthe substrate in a 1:1 mixture of Py*HCl/NMP at 190° C. for 10 h. Afterpurification by column chromatography, 7.8 g (37% yield) of Example 4(free base) was obtained. It was treated with 10% HCl solution in EtOHto give 8.4 g of pure Example 4.

Experimental

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Proton nuclear magnetic resonance spectra wereobtained on a Bruker ARX 400 spectrometer at 400 MHz. The solvent peakwas used as the reference peak for proton spectra. The progress ofreactions was controlled by TLC or/and LCMS analysis.

4-Hydroxy-1-indanone (76)

Anhydrous aluminum chloride (550 g, 4.135 mol, 4 eq) and sodium chloride(105 g, 1.795 mol, 2.7 eq) were mixed and heated to 150° C.Dihydrocoumarin (75) (100 g, 675.7 mol, 1 eq) was added slowlymaintaining the internal temperature between 150-160° C. After theaddition, the temperature was increased to 200° C. and the reactionmixture stirred for 1.5 h, and while hot, poured into a porcelain dishto cool. The solidified mass was broken and added to a vigorouslystirred mixture of ice and water (4 L) containing 400 mL conc. HCl. Theresulting suspension was stirred for 1 h, filtered, washed with waterand dried to give 85 g (85%) of crude 4-hydroxy-1-indanone (76) as agrey solid. It was sufficiently pure to be used in the next step.

Trifluoromethanesulfonic acid 1-oxoindan-4-yl ester (77)

Trifluoromethanesulfonic acid anhydride (72.3 mL, 429.7 mmol, 1.2 eq)was added to a suspension of 4-hydroxy-1-indanone (77) (53 g, 358.1mmol, 1 eq) in dry CH₂Cl₂ (450 mL) and pyridine (87 mL, 1074 mmol, 3eq), while maintaining the internal temperature below 5° C. The reactionmixture was allowed to warm up to room temperature and the stirringcontinued for 1 h. CH₂Cl₂ (300 mL) and water (100 mL) were added to thereaction mixture, the organic layer was separated, washed subsequentlywith 2% HCl solution (3×100 mL), and saturated NaHCO₃ (2×100 mL), anddried over Na₂SO₄. CH₂Cl₂ was evaporated out and the crude productpurified by flash chromatography (5% to 10% EtOAc in Hexanes) to give 90g (90%) of pure 77 as a brown liquid.

¹H-NMR (DMSO-d₆): 2.76 (t, 2H), 3.19 (t, 2H), 7.65 (dd, 1H), 7.94 (2d,2H).

Trifluoromethanesulfonic acid 4-methoxy-2-nitrophenyl ester (78)

Trifluoromethanesulfonic acid anhydride (60 mL, 350 mmol, 1.2 eq) wasadded to a solution of 4-methoxy-2-nitrophenol (78) (50.57 g, 298.9mmol, 1 eq) in dry CH₂Cl₂ (550 mL) and pyridine (72.5 mL, 897 mmol, 3eq), while maintaining the internal temperature below 5° C. The reactionmixture was allowed to warm to room temperature and stirred overnight.Water (100 mL) was added to the reaction mixture, the organic layer wasseparated, washed subsequently with solutions of 7% HCl solution, thensaturated NaHCO₃, and dried over Na₂SO₄. The CH₂Cl₂ solution wasfiltered through a pad of SiO₂ and the solvent removed in vacuum to give89 g (99%) of the pure triflate 79 as a pale yellow liquid.

¹H-NMR (DMSO-d₆): 3.92 (s, 3H), 7.49 (dd, 1H), 7.71 (d, 1H), 7.81 (d,1H).

4-(4-Methoxy-2-nitrophenyl)-indan-1-one (81)

A mixture consisting of potassium acetate (29.44 g, 0.3 mol, 1.5 eq),triflate (77) (61.44 g, 0.22 mol, 1.1 eq), bis(pinacolo)diborane (60.94g, 0.24 mol, 1.2 eq), PPh₃ (3.15 g, 0.012 mol, 0.06 eq), andPdCl₂(PPh₃)₂ (4.21 g, 0.006 mol, 0.03 eq) in degassed toluene (2 L) wasrefluxed under argon atmosphere overnight. The triflate (79) (60.24 g,0.2 mol, 1 eq), Pd(PPh₃)₄ (11.55 g, 0.01 mol, 0.05 eq), and 2M Na₂CO₃(800 mL) were added consecutively to the same flask under a rapid flowof argon, and the resulting solution was refluxed overnight. Water (200mL) and EtOAc (500 mL) were added, the aqueous layer was separated andextracted with EtOAc (2×500 mL) and the combined extracts dried overNa₂SO₄. The solvent was evaporated at reduced pressure and the crudeproduct purified by column chromatography (20% EtOAc in hexanes) to give44 g (80%) of the biphenyl intermediate (81) as a yellow solid.

8-Methoxy-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (82)

A solution of 4-(4-methoxy-2-nitrophenyl)-indan-1-one (81) (42 g, 148.4mmol, 1 eq) and triphenylphosphine (116 g, 445 mmol, 3 eq) ino-dichlorobenzene (400 mL) was refluxed with vigorous stirring for 4 h,then cooled to 0° C. Diethyl ether (4 L) was added and the precipitatefiltered off, washed with diethyl ether (3×100 mL) and dried to give 17g (46%) of (82). The filtrate was evaporated out and the residue washedwith cold MeOH (5×100 ml) to give an additional 10 g (27%) of thecarbazole (82). Total yield 27 g (73%).

¹H-NMR (DMSO-d₆): 2.74 (m, 2H), 3.50 (m, 2H), 3.88 (s, 3H), 6.90 (d,1H), 7.08 (s, 1H), 7.47 (d, 1H), 7.58 (d, 1H), 7.97 (d, 1H), 11.79 (s,1H).

9-Acetyl-8-methoxy-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (83)

8-Methoxy-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (82) (27.8 g,110.8 mmol, 1 eq) was dissolved in dry dichloromethane (300 mL).Anhydrous AlCl₃ (29.5 g, 221.5 mmol, 2 eq) was added in portions withstirring and cooling, followed by the dropwise addition of AcCl (24 mL,332.4 mmol, 3 eq). The reaction mixture was stirred at about 5° C. for24 h and poured into ice water (violent foaming should be avoided). Theprecipitated orange solid was filtered off, washed with water (10×100mL), CH₂Cl₂ (3×50 mL), acetone (3×50 mL) to give 26 g (80%) of9-acetyl-8-methoxy-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (83).

9-Acetyl-8-methoxy-1,2-dihydro-6-(3-dimethylaminopropyl)-cyclopenta[c]carbazole-3-one (84)

NaH (6.88 g of 60%, 171.95 mmol, 2.5 eq) was added to a suspension of9-acetyl-8-methoxy-1,2-dihydro-6H-cyclopenta[c]carbazole-3-one (83) (26g, 68.78 mmol, 1 eq) in dry CH₂Cl₂ (300 mL), and the mixture was stirredat room temperature for 20-30 min until the evolution of hydrogenceased. 3-Dimethylaminopropylchloride hydrochloride (16.3 g, 103.16mmol, 1.5 eq) was added portionwise under nitrogen. The reaction mixturewas stirred for 30 min at room temperature, then at 60° C. for 24 h andpoured into ice water (4 L). The aqueous solution was acidified to pHabout 2 with conc. HCl and the unreacted starting material extractedwith EtOAc/THF=3/1 mixture. Upon adjusting the pH of the water layer toabout 9, the product was extracted with 1:1 EtOAc:THF mixture (10×500mL). The combined extracts were washed with brine, dried over Na₂SO₄ andsolvent evaporated out to afford 22 g (68%) of crude carbazole (84),reasonable pure by LC/MS. It was used in the next step without furtherpurification.

¹H-NMR (DMSO-d₆): 1.88 (t, 2H), 2.13 (s, 6H), 2.16 (t, 2H), 2.62 (s,3H), 2.75 (m, 2H), 3.47 (m, 2H), 4.01 (s, 3H), 4.52 (t, 2H), 7.32 (s,2H), 7.65 (dd, 4H), 8.32 (s, 2H).

9-Acetyl-8-hydroxy-1,2-dihydro-6-(3-dimethylaminopropyl)-cyclopenta[c]carbazole-3-one

A solution of9-acetyl-8-methoxy-1,2-dihydro-6-(3-dimethylaminopropyl)-cyclopenta[c]carbazole-3-one(84) (22 g, 58.2 mmol, 1 eq) and pyridine hydrochloride (134.4 g, 1164mmol, 20 eq) in NMP (150 mL) was refluxed for 10 h. The reaction mixturewas cooled to room temperature and poured into the 10% aqueous K₂CO₃solution (3 L). The precipitated dark green solid was filtered off,washed with water (5×100 mL) and dried. The crude product was purifiedby column chromatography (eluent 10%-20% MeOH in EtOAc) to give 7.81(37%) of pure9-acetyl-8-hydroxy-1,2-dihydro-6-(3-dimethylaminopropyl)-cyclopenta[c]carbazole-3-oneas a pale yellow solid.

MS(ESI): m/z=363.3 [M+H]⁺

NMR ¹H (DMSO): 1.86 (t, 2H), 2.17 (s, 6H), 2.20 (t, 2H), 2.79 (m, 2H),2.81 (s, 3H), 3.52 (m, 2H), 4.43 (t, 2H), 7.15 (s, 2H), 7.66 (dd, 4H),8.50 (s, 2H), 12.71 (s, 1H).

Example 4

9-Acetyl-8-hydroxy-1,2-dihydro-6-(3-dimethylaminopropyl)-cyclopenta[c]carbazole-3-one(7.81 g, 21.46 mmol, 1 eq) was dissolved in a mixture of water (20 mL)and 10% HCl solution (20 mL) in ethanol (200 mL), and the homogeneoussolution evaporated to dryness. The residue was dried in vacuumovernight to give 8.47 g of Example 4 as a gray solid.

Purity: 99.2% by HPLC; MS(ESI): m/z=363.3 [M−HCl+H]⁺; m.p.=241.3° C.(Decomposition); ¹H-NMR (DMSO-d₆): 2.15 t (2H), 2.68 s (3H), 2.69 s(3H), 2.80 m (2H), 2.82 s (3H), 3.11 m (2H), 3.55 m (2H), 4.53 t (2H),7.29 s (2H), 7.73 dd (4H), 8.52 s (2H), 11.00 s (1H), 12.76 s (1H).

Compounds of structural formula (II) are prepared similarly to compoundsof structural formula (I), and include for example,

Additional carbazole compounds of the present invention are:

A compound useful in the methods of the present invention has astructure:

Additional examples having a structural formula (I) include, but are notlimited to:

Compounds of the present invention therefore include, but are notlimited to:

The potency of a carbazole compound is determined by measuring anability of the compound to activate p53. In particular, a p53 responsiveluciferase reporter cell line is used to identify compounds capable ofactivating p53. The activation of p53 is reported as the EC₅₀ value,which is the effective concentration of the compound needed to increasep53 activity by 50% over a baseline p53 activity.

The p53 activation assay for determination of the EC₅₀ value wasperformed as follows:

Definitions and Terminology

ConALuc: p53 responsive luciferase reporter construct

DMSO: Dimethyl sulfoxide

FBS: fetal bovine serum

Equipment

96 well luminometer-fluoroscan (e.g., Fluoroscan, LabSystems, Inc,settings-program, integration time is 0.1 sec, PMT voltage is 1000, andzero lag time

Multichannel pipette 50-300 uL range

96 well plates

Materials

Reporter cell lines:

HT1080-L (human fibrosarcoma cells with ConALuc reporter)

RCC45ConALuc (human renal cell carcinoma cell line with ConALucreporter)

Standard DMEM Medium

Standard RPMI Medium

Pen/strep 100×

Trypsin-EDTA 10×-dilute to 1× in sterile PBS

PBS

Bright-Glo luciferase assay system (Fisher PR-E2620)

9-aminoacridine (9aa) 20 mM in DMSO (Sigma A38401), p53 activator(positive control)

Compound 100 20 mM in DMSO (Chembridge), p53 activator (positivecontrol)

DMSO (Fisher D128-500) (negative control)

Method

1. Two types of standard cells were used, either HT1080-L orRCC45ConALuc cells. Both cell lines stably express a p53 responsiveluciferase reporter construct.

2. HT1080-L cells were grown in DMEM medium containing 10% FBS.RCC45ConALuc cells were grown in RPMI medium containing 10% FBS(Pen/Strep can be added to a final concentration of 1%, if desired).Both cell lines were grown in a humidified incubator at 37° C. with 5%CO₂. For normal culturing, both cell lines were split using 1×trypsin-EDTA at a ratio of 1:20 for HT1080-L and 1:5 for RCC45ConALuccells every 3-4 days (when cells are 80-90% confluent).

3. A day before the experiment, the cells are trypsinized for about 5minutes in 1× tripsin/EDTA solution in 37° C. incubator and plated in 96well plates. HT1080-L cells were seeded at a density of 1×10⁴/well instandard DMEM medium in a volume of 50 μl. RCC45ConALuc were seeded at adensity of 2×10⁴/well in the volume of 50 uL in standard RPMI medium.

4. The next day, various carbazole compounds were prepared by dilutionof stock solutions in standard DMEM medium such that the cells weretreated with the final chemical concentrations in Table 1 below. Stocksolutions were made up in DMSO. Typically, chemicals were made up as 20mM stock solutions. However, this concentration is dependent on thesolubility of a given compound and therefore actual stock concentrationswere noted at time of experiment (e.g., less soluble compounds can havea stock concentration of 5 or 10 mM).

5. Each tested compound used two rows of a 96-well plate, thus fourcompounds (e.g., W, X, Y, Z) were tested in one plate simultaneously. Inaddition to test compounds, each plate included positive and negativecontrols. As positive control, 9aa was used at a dose of 3 μM. As anegative control, DMSO was used in final concentration 0.1%.

TABLE 1 Scheme of an experimental plate with final concentrations ofchemicals Library compound (Cpd W, X, Y, Z) (uM) Neg Control Pos.Control

Cpd W DMSO 3 uM 9aa 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd WDMSO 3 uM 9aa 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd X DMSO3 uM 9aa 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd X DMSO 3 uM9aa 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd Y DMSO 3 uM 9aa0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd Y DMSO 3 uM 9aa 0.0390.078 0.156 0.313 0.625 1.25 2.5 5 10 20 Cpd Z DMSO 3 uM 9aa 0.039 0.0780.156 0.313 0.625 1.25 2.5 5 10 20 Cpd Z DMSO 3 uM 9aa 0.039 0.078 0.1560.313 0.625 1.25 2.5 5 10 20

1. Dilutions of chemicals added to the plate were made up as 2×concentrations in standard DMEM and added to corresponding well in avolume of 50 μl.

2. Chemicals were diluted in standard DMEM by two time serial dilutionsstarting from 40 μM (2× of highest concentration, e.g., 20 μM). For onecell line, the minimum volume was 125 μL of 2× working solution ofchemical of each concentration in standard DMEM.

3. In addition to a positive control added in one concentration in eachplate, a positive control of Compound 100, or the most active compoundon each stage of screening, was added to each assay run in a fullconcentration range to ensure proper and robust assay performance(estimated by comparison of dose response curves of p53 activation amongruns).

4. Sixteen hours following compound addition, all wells with the highestconcentrations of compounds are checked microscopically for the presenceof toxic effect, because death of cells caused by compounds may notallow detecting luciferase activity. If cytotoxicity is evident, otherdoses of the same compounds were checked for the presence of toxicity.The lowest dose causing toxic effect was recorded. In case cytotoxicitywas observed at 3-4 lowest compound doses (in a current dose scheme at0.3 and lower uM), the compound was retested separately in a lowerconcentration range, allowing at least 4 two-fold-different doses ofcompound to be tested without signs of cytotoxicity).

5. After microscopic examination, 15 μl of Bright Glo luciferase assaysystem were added to each well and after 5 min incubation at RT, theplates were read on a 96 well plate luminometer with a measurement timeof 0.1 sec. A shaking step was included before measurement.

6. The folds of luciferase activation for each concentration of eachchemical were calculated by dividing the detected luciferase activity ateach chemical concentration by the average of the luciferase activityfor the DMSO control on the same plate. The folds then were plottedversus the chemical concentration to determine whether compounds areactive. From the data, both the maximum fold activation and the Emax(concentration causing maximal p53 activation) were determined.

7. For each compound, this assay is repeated two additional times. Oncethree runs were complete, the raw data was used to calculate the EC₅₀value.

An assay was considered invalid when:

a difference between two duplicates increases 10% for more than 10% ofreadings;

death of DMSO treated cells is observed;

less than 3 times luciferase induction in cells treated with positivecontrol (Compound 3a, 0.4 uM) versus luciferase activity of DMSO treatedcells;

inability to get dose-dependent curve of luciferase induction withpositive control (e.g. Compound 100, 0.03-20 uM).

The following are nonlimiting examples of EC₅₀ values for variouscarbazole compounds useful in the method of the present invention:

EC₅₀ value (p53 activation, Compound μM) Compound 100 1.30 Example 210.83 Example 22 0.53 Compound 3a 0.29 Compound 3b 0.49 Compound 7d 0.64Example 23 0.88 Example 24 0.78 Example 25 0.88 Example 2 0.64 Example27 0.54 Example 15 0.03 Compound 19a 0.40 Compound 3e 0.78 Example 70.37 Example 4 0.07 Compound 18c-1 0.08 Compound 18c-2 0.10 Compound 19e0.07 Example 13 0.22 Example 14 0.05 Example 6 0.24 Example 17 0.04Example 18 0.09 Example 38 0.12

Compounds and pharmaceutical compositions of the present inventioninclude those wherein the active ingredient is administered in atherapeutically effective amount to achieve its intended purpose. A“therapeutically effective amount” refers to that amount of a presentcarbazole compound that results in achieving the desired effect.Toxicity and therapeutic efficacy of the carbazole compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, which is expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred. The data obtained from such data can be used informulating a dosage range for use in humans. The dosage preferably lieswithin a range of circulating compound concentrations that include theED₅₀ with little or no toxicity. The dosage can vary within this rangedepending upon the dosage form employed, and the route of administrationutilized. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

A therapeutically effective amount of a carbazole compound of structuralformula (I) or (II) required for use in therapy varies with the natureof the condition being treated, the length of time that activity isdesired, and the age and the condition of the patient, and ultimately isdetermined by the attendant physician. Dosage amounts and intervals canbe adjusted individually to provide plasma levels of the carbazolecompound that are sufficient to maintain the desired therapeuticeffects. The desired dose conveniently can be administered in a singledose, or as multiple doses administered at appropriate intervals, forexample as one, two, three, four or more subdoses per day. Multipledoses often are desired, or required. For example, a present carbazolecompound can be administered at a frequency of: four doses delivered asone dose per day at four-day intervals (q4d×4); four doses delivered asone dose per day at three-day intervals (q3d×4); one dose delivered perday at five-day intervals (qd×5); one dose per week for three weeks(qwk3); five daily doses, with two days rest, and another five dailydoses (5/2/5); or, any dose regimen determined to be appropriate for thecircumstance.

The dosage of a composition containing a carbazole compound ofstructural formula (I) or (II), or a composition containing the same,can be from about 1 ng/kg to about 200 mg/kg, about 1 μg/kg to about 100mg/kg, or about 1 mg/kg to about 50 mg/kg. The dosage of a compositioncan be at any dosage including, but not limited to, about 1 μg/kg. Thedosage of a composition may be at any dosage including, but not limitedto, about 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg,125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg,450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg,775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, or 200 mg/kg. The above dosages are exemplary of the averagecase, but there can be individual instances in which higher or lowerdosages are merited, and such are within the scope of this invention.

When administered in combination with other therapeutics, a presentcarbazole compound may be administered at relatively lower dosages. Inaddition, the use of targeting agents may allow the necessary dosage tobe relatively low. Certain compounds may be administered at relativelyhigh dosages due to factors including, but not limited to, low toxicityand high clearance.

For human use, a compound of structural formula (I) or (II) can beadministered alone, but generally is administered in admixture with apharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present invention can beformulated in a conventional manner using one or more physiologicallyacceptable carrier comprising excipients and auxiliaries that facilitateprocessing of compounds of formula (I) or (II) into pharmaceuticalpreparations.

The present carbazole compounds can be administered simultaneously ormetronomically with other anti-cancer treatments, such as chemotherapyand/or radiation therapy. The term “simultaneous” or “simultaneously”means that the other anti-cancer treatment and the carbazole compoundare administered within 6 hours, 3 hours or less, of each other. Theterm “metronomically” means the administration of the other anti-cancertreatments at times different from the anti-cancer treatments and at acertain frequency relative to repeat administration and/or theanti-cancer treatment regiment.

The carbazole compound, or compositions containing the carbazolecompound, can be administered in any manner including, but not limitedto, orally, parenterally, sublingually, transdermally, rectally,transmucosally, topically, via inhalation, via buccal administration, orcombinations thereof. Parenteral administration includes, but is notlimited to, intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, and intraarticular. The carbazole compoundalso can be administered in the form of an implant, which allows a slowrelease of the compound, as well as a slow controlled i.v. infusion.

The carbazole compounds of the present invention can be used to treat avariety of diseases and conditions. For example, compounds of thepresent invention can be used in combination with radiation and/or achemotherapeutic agent in the treatment of cancers. For example, thecarbazole compounds can be used to enhance treatment of tumors that arecustomarily treated with an antimetabolite, e.g., methotrexate or5-fluorouracil (5-FU).

Use of carbazole compounds of the present invention can result inpartial or complete regression of cancer cells, i.e., the partial orcomplete disappearance of such cells from the cell population. Forexample, a method of the invention can be used to slow the rate of tumorgrowth, decrease the size or number of tumors, or to induce partial orcomplete tumor regression.

A present carbazole compound can be used for treating a disease orcondition in vivo by administration to an individual in need thereof.The disease or condition can be a cancer. A variety of cancers can betreated including, but not limited to: carcinomas, including bladder(including accelerated and metastic bladder cancer), breast, colon(including colorectal cancer), kidney, liver, lung (including small andnon-small cell lung cancer and lung adenocarcinoma), ovary, prostate,testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas(including exocrine pancreatic carcinoma), esophagus, stomach, gallbladder, cervix, thyroid, renal, and skin (including squamous cellcarcinoma); hematopoietic tumors of lymphoid lineage, includingleukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkinslymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkettslymphoma, hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias, myelodysplastic syndrome, myeloidleukemia, and promyelocytic leukemia; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscarcoma, and osteosarcoma; and other tumors, includingmelanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroidfollicular cancer, teratocarcinoma, renal cell carcinoma (RCC),pancreatic cancer, myeloma, myeloid and lymphoblastic leukemia,neuroblastoma, and glioblastoma.

Transformation induced by tax of HTLV, a causative agent of human adultT-lymphoblastic leukemia (ATL), may share the same molecular targetsinvolved in RCC. For example, NF-κB is constitutively active intax-transformed cells. Similar to RCC, p53 activity is inhibited throughactivation of NF-κB in tax-transformed cells and p53 inhibition does notinvolve sequestering of p300. Based on the shared mechanism of p53activation, the compositions may also be used to treat HTLV-inducedleukemia. Regardless of their p53 status, the majority of human cancershave constitutively hyperactivated NF-κB. The composition also iscapable of inhibiting NF-κB by reprogramming transactivation NF-κBcomplexes into transrepression complexes, which can be used fortreatment of any tumor regardless of their p53 status. The compositionsfurther can be used for treating HIV infections because HIV LTRs arestrongly dependent on NF-κB activity.

The composition also can be used as an adjuvant therapy to overcomeanti-cancer drug resistance that can be caused by constitutive NF-κBactivation. The anti-cancer drug can be a chemotherapeutic or radiation,as described herein.

One method of the present invention comprises administration of atherapeutically effective amount of a present carbazole compound incombination with a chemotherapeutic agent that can effect single- ordouble-strand DNA breaks or that can block DNA replication or cellproliferation. Alternatively, a method of the present inventioncomprises administration of a therapeutically effective amount of atleast one present carbazole compound in combination with therapies thatinclude use of an antibody, e.g., herceptin, that has activity ininhibiting the proliferation of cancer cells. Accordingly, cancers, forexample, colorectal cancers, head and neck cancers, pancreatic cancers,breast cancers, gastric cancers, bladder cancers, vulvar cancers,leukemias, lymphomas, melanomas, renal cell carcinomas, ovarian cancers,brain tumors, osteosarcomas, and lung carcinomas, are susceptible toenhanced treatment by administration of a present carbazole incombination with a chemotherapeutic agent or an antibody.

Cancers treatable by the present invention also include solid tumors,i.e., carcinomas and sarcomas. Carcinomas include malignant neoplasmsderived from epithelial cells which infiltrate (i.e., invade)surrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or from tissues that formrecognizable glandular structures. Another broad category of cancersincludes sarcomas, which are tumors whose cells are embedded in afibrillar or homogeneous substance, like embryonic connective tissue.The present invention also enables treatment of cancers of the myeloidor lymphoid systems, including leukemias, lymphomas, and other cancersthat typically are not present as a tumor mass, but are distributed inthe vascular or lymphoreticular systems.

Additional forms of cancer treatable by the present carbazole compoundsinclude, for example, adult and pediatric oncology, growth of solidtumors/malignancies, myxoid and round cell carcinoma, locally advancedtumors, metastatic cancer, human soft tissue sarcomas, including Ewing'ssarcoma, cancer metastases, including lymphatic metastases, squamouscell carcinoma, particularly of the head and neck, esophageal squamouscell carcinoma, oral carcinoma, blood cell malignancies, includingmultiple myeloma, leukemias, including acute lymphocytic leukemia, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, and hairy cell leukemia, effusion lymphomas (bodycavity based lymphomas), thymic lymphoma lung cancer (including smallcell carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producingtumors, nonsmall cell cancers, breast cancer, including small cellcarcinoma and ductal carcinoma), gastrointestinal cancers (includingstomach cancer, colon cancer, colorectal cancer, and polyps associatedwith colorectal neoplasia), pancreatic cancer, liver cancer, urologicalcancers (including bladder cancer, such as primary superficial bladdertumors, invasive transitional cell carcinoma of the bladder, andmuscle-invasive bladder cancer), prostate cancer, malignancies of thefemale genital tract (including ovarian carcinoma, primary peritonealepithelial neoplasms, cervical carcinoma, uterine endometrial cancers,vaginal cancer, cancer of the vulva, uterine cancer and solid tumors inthe ovarian follicle), malignancies of the male genital tract (includingtesticular cancer and penile cancer), kidney cancer (including renalcell carcinoma, brain cancer (including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, and metastatic tumorcell invasion in the central nervous system), bone cancers (includingosteomas and osteosarcomas), skin cancers (including malignant melanoma,tumor progression of human skin keratinocytes, and squamous cellcancer), thyroid cancer, retinoblastoma, neuroblastoma, peritonealeffusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gallbladder cancer, trophoblastic neoplasms, hemangiopericytoma, andKaposi's sarcoma. Accordingly, administration of a present carbazolecompound is expected to enhance treatment regimens.

Compounds of the present invention also can potentiate the efficacy ofdrugs in the treatment of inflammatory diseases. The present carbazolecompounds are potent inhibitors of NF-κB response, which involves thesuppression of expression/secretion of pro-inflammatory NF-κB targets,such as TNF, IL-1, IL-6, IL-8, and many others. Therefore, theinhibition of NF-κB by a present carbazole would lead to the suppressionof local and systemic inflammatory reactions. Quinacrine, which ishypothesized to act by a mechanism very similar to the presentcarbazole, was widely used as an anti-inflammatory agent for thetreatment of autoimmune diseases and chronic inflammation.

Examples of diseases that can benefit from combination therapy withcompounds suitable for the method of the present invention arerheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, andsystemic lupus erythematosus (SLE). Treatment of arthritis, Wegener'sgranulomatosis, and SLE often involves the use of immunosuppressivetherapies, such as ionizing radiation, methotrexate, andcyclophosphamide. Such treatments typically induce, either directly orindirectly, DNA damage. Inhibition of NF-κB and/or activation of p53within the offending immune cells render the cells more sensitive tocontrol by these standard treatments. Psoriasis and vitiligo commonlyare treated with ultraviolet radiation (UV) in combination withpsoralen. The present carbazole compounds induce the killing effect ofUV and psoralen, and increase the therapeutic index of this treatmentregimen. In general, the carbazole compounds useful in methods of thepresent invention potentiate control of inflammatory disease cells whenin combination with currently used immunosuppressive drugs.

In addition to the above conditions, the present invention also can beused in methods of treating conditions such as, but not limited to,atherosclerosis, restenosis, vasculitis, nephritis, retinopathy, renaldisease, proliferative skin disorders, psoriasis, keloid scarring,actinic keratosis, Stevens-Johnson Syndrome, rheumatoid arthritis (RA),systemic-onset juvenile chronic arthritis (JCA), osteoporosis, systemiclupus erythmatosis, hyperproliferative diseases of the eye includingepithelial down growth, proliferative vitreoretinopathy (PVR), diabeticretropathy, Hemangio-proliferative diseases, ichthyosis, and papillomas.

Carbazoles of the present invention also exhibit antimicrobial activity,e.g., agent Germ-positive and Germ-negative bacteria, includingSalmonella; antiprotozoan activity; and antiviral activity.

As appreciated by persons skilled in the art, additional active orancillary agents can be used in the methods described herein. Referenceherein to treatment also extends to prophylaxis, as well as to treatmentof established diseases or symptoms.

The present invention can be applied to cell populations ex vivo. Forexample, the present carbazole compounds can be used ex vivo todetermine the optimal schedule and/or dosing of administration of thepresent carbazole compound for a given indication, cell type, patient,and other parameter. Information gleaned from such use can be used forexperimental purposes or in the clinic to set protocol for in vivotreatment. Other ex vivo uses for which the invention is suited areapparent to those skilled in the art.

A present carbazole compound also can be administered in combinationwith radiation. Diseases that are treatable with electromagneticradiation include neoplastic diseases, benign and malignant tumors, andcancerous cells.

Electromagnetic radiation treatment of other diseases not listed hereinalso is contemplated by the present invention. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹² to 10⁻¹⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1 mm), and microwave radiation (1 mm to 30cm).

Many cancer treatment protocols currently employ radiosensitizersactivated by electromagnetic radiation, e.g., X-rays. Examples ofX-ray-activated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FUdR), hydroxyurea, cis-platin, and therapeutically effective analogsand derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

Radiosensitizers can be administered in conjunction with atherapeutically effective amount of one or more compounds in addition toa present carbazole compound, such compounds including, but not limitedto, compounds that promote the incorporation of radiosensitizers to thetarget cells, compounds that control the flow of therapeutics,nutrients, and/or oxygen to the target cells, chemotherapeutic agentsthat act on the tumor with or without additional radiation, or othertherapeutically effective compounds for treating cancer or otherdisease. Examples of additional therapeutic agents that can be used inconjunction with radiosensitizers include, but are not limited to,5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red celltransfusions, perfluorocarbons (e.g., FLUOSOLW®-DA), 2,3-DPG, BW12C,calcium channel blockers, pentoxifylline, antiangiogenesis compounds,hydralazine, and L-BSO.

The chemotherapeutic agent can be any pharmacological agent or compoundthat induces apoptosis. The pharmacological agent or compound can be,for example, a small organic molecule, peptide, polypeptide, nucleicacid, or antibody. Chemotherapeutic agents that can be used include, butare not limited to, alkylating agents, antimetabolites, hormones andantagonists thereof, natural products and their derivatives,radioisotopes, antibodies, as well as natural products, and combinationsthereof. For example, a carbazole compound of the present invention canbe administered with antibiotics, such as doxorubicin and otheranthracycline analogs, nitrogen mustards, such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cis-platin, hydroxyurea,taxol and its natural and synthetic derivatives, and the like. Asanother example, in the case of mixed tumors, such as adenocarcinoma ofthe breast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the compound can be administered inconjunction with leuprolide or goserelin (synthetic peptide analogs ofLH-RH). Other antineoplastic protocols include the use of an inhibitorcompound with another treatment modality, e.g., surgery or radiation,also referred to herein as “adjunct anti-neoplastic modalities.”Additional chemotherapeutic agents useful in the invention includehormones and antagonists thereof, radioisotopes, antibodies, naturalproducts, and combinations thereof.

Examples of chemotherapeutic agents useful for the method of the presentinvention are listed in the following table.

TABLE 1 Alkylating agents Natural products Nitrogen mustards Antimitoticdrugs mechlorethamine Taxanes cyclophosphamide paclitaxel ifosfamideVinca alkaloids melphalan vinblastine (VLB) chlorambucil vincristineuracil mustard vinorelbine temozolomide vindesine NitrosoureasTaxotere ® (docetaxel) carmustine (BCNU) estramustine lomustine (CCNU)estramustine phosphate semustine (methyl-CCNU) Epipodophylotoxinschlormethine etoposide streptozocin teniposideEthylenimine/Methyl-melamine Antibiotics triethylenemelamine (TEM)actimomycin D triethylene thiophosphoramide daunomycin (rubidomycin)(thiotepa) doxorubicin (adriamycin) hexamethylmelaminemitoxantroneidarubicin (HMM, altretamine) bleomycin Alkyl sulfonatessplicamycin (mithramycin) busulfan mitromycin-C pipobroman dactinomycinTriazines aphidicolin dacarbazine (DTIC) epirubicin Antimetabolitesidarubicin Folic Acid analogs daunorubicin methotrexate mithramycintrimetrexate deoxy co-formycin pemetrexed Enzymes (Multi-targetedantifolate) L-asparaginase Pyrimidine analogs L-arginase 5-fluorouracilRadiosensitizers fluorodeoxyuridine metronidazole gemcitabinemisonidazole cytosine arabinoside desmethylmisonidazole (AraC,cytarabine) pimonidazole 5-azacytidine etanidazole2,2′-difluorodeoxy-cytidine nimorazole floxuridine RSU 1069 pentostatineEO9 Purine analogs RB 6145 6-mercaptopurine Nonsteroidal antiandrogens6-thioguanine SR4233 azathioprine flutamide 2′-deoxycoformycinnicotinamide (pentostatin) 5-bromodeozyuridineerythrohydroxynonyl-adenine (EHNA) 5-iododeoxyuridine fludarabinephosphate bromodeoxycytidine 2-chlorodeoxyadenosine Miscellaneous agents(cladribine, 2-CdA) Platinium coordination complexes Type ITopoisomerase Inhibitors cisplatin camptothecin carboplatin topotecanoxaliplatin irinotecan anthracenedione Biological response modifiersmitoxantrone G-CSF Substituted urea GM-CSF hydroxyurea DifferentiationAgents Methylhydrazine derivatives retinoic acid derivativesN-methylhydrazine (MIH) Hormones and antagonists procarbazineAdrenocorticosteroids/antagonists Adrenocortical suppressant prednisoneand equivalents mitotane (o,p′-DDD) dexamethasone ainoglutethimideainoglutethimide Cytokines Progestins interferon (α, β, γ)hydroxyprogesterone caproate interleukin-2 medroxyprogesterone acetatePhotosensitizers megestrol acetate hematoporphyrin derivatives EstrogensPHOTOFRIN ® diethylstilbestrol benzoporphyrin derivatives ethynylestradiol/equivalents Npe6 Antiestrogen tin etioporphyrin (SnET2)tamoxifen pheoboride-a Androgens bacteriochlorophyll-a testosteronepropionate naphthalocyanines fluoxymesterone/equivalents phthalocyaninesAntiandrogens zinc phthalocyanines flutamide Radiationgonadotropin-releasing X-ray hormone analogs ultraviolet lightleuprolide gamma radiation visible light infrared radiation microwaveradiation

Examples of chemotherapeutic agents that are particularly useful inconjunction with radiosensitizers include, for example, camptothecin,carboplatin, cis-platin, daunorubicin, doxorubicin, interferon (alpha,beta, gamma), irinotecan, hydroxyurea, chlorambucil, 5-fluorouracil(5-FU), methotrexate, 2-chloroadenosine, fludarabine, azacytidine,gemcitabine, pemetrexed, interleukin 2, irinotecan, docetaxel,paclitaxel, topotecan, CPT-11, anastrazole, letrazole, capecitabine,reloxafine, cyclophosphamide, ifosamide, droloxafine, andtherapeutically effective analogs and derivatives of the same.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395),colchicines (NSC 757), colchicines derivatives (e.g., NSC 33410),dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC332598), paclitaxel (NSC 125973), TAXOL® derivatives (e.g., NSC 608832),thiocolchicine NSC 361792), trityl cysteine (NSC 83265), vinblastinesulfate (NSC 49842), vincristine sulfate (NSC 67574), natural andsynthetic epothilones including but not limited to epothilone A,eopthilone B, and discodermolide (see Service, (1996) Science, 274:2009)estramustine, nocodazole, MAP4, and the like. Examples of such agentsare also described in Bulinski (1997) J. Cell Sci. 110:3055 3064; Panda(1997) Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) CancerRes. 57:3344-3346; Nicolaou (1997) Nature 397:268-272; Vasquez (1997)Mol. Biol. Cell. 8:973-985; and Panda (1996) J. Biol. Chem271:29807-29812.

Cytostatic agents that may be used include, but are not limited to,hormones and steroids (including synthetic analogs):17-α-ethinylestadiol, diethylstilbestrol, testosterone, prednisone,fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, chlorotrianisene, hydroxyprogesterone, aminogluthimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, zoladex.

Other cytostatic agents are antiangiogenics such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU668.Anti-Her2 antibodies also may be utilized. An EGFR inhibitor is EKB-569(an irreversible inhibitor). Also included are antibody C225immunospecific for the EGFR and Src inhibitors.

Also suitable for use as a cytostatic agent is CASODEX® (bicalutamide,Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen TAMOXIFEN® which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include epidermal growth factor inhibitors, Her-2 inhibitors,MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Srckinase inhibitors, and PDGF inhibitors.

Due to their unique ability to induce apoptosis in tumor cells, TNFfamily members are considered to be potential anticancerpharmaceuticals. However, many tumor cells escape proapoptotic acid ofdeath ligands, thereby reducing the use of these agents to deathligand-sensitive cancers and allowing the tumor to escape host immuneresponse. The use of an inhibitor of NF-κB may be used to sensitizetumor cells to the killing of a death ligand, such as a TNF polypeptide.

The TNF polypeptide can be a member of the TNF superfamily of ligands.Representative examples of TNF polypeptides include, but are not limitedto, NGF, CD40L, CD137L/4-1BBL, TNF-α, CD134L/OX40L, CD27L/CD70,FasL/CD95, CD30L, TNF-β/LT-α, LT-β, and TRAIL. Members of the TNFsuperfamily are natural proteins that are implicated in the maintenanceand function of the immune system and that can trigger apoptosis. TheTNF polypeptide may be TRAIL, which induces apoptosis mainly in tumorbut not in normal cells. The activity of these so-called “death ligands”is believed to be mediated by binding with members of the TNF receptorfamily, which contain structurally similar death domains in theirintracellular portions. Ligation of these receptors, specific for eachdeath ligand, trigger activation of a cascade of events resulting incaspase activation. Representative examples of TNF-R receptors bound bythe TNF polypeptides include, but are not limited to, LNGFR/p75, CD40,CD137/4-1BB/ILA, TNFRI/p55/CD120a, TNFRII/p75/CD120b, CD134/OX40/ACT35,CD27, Fas/CD95/APO-1, CD30/Ki-1, LT-β R, DR3, DR4, DR5, DcR1/TRID, TR2,GITR and osteoprotegerin.

It also contemplated that other agents can be used in the place of theTNF polypeptide. For example, an antibody that mimics the activity of aTNF polypeptide can be used. Representative examples of such antibodiesinclude, but are not limited to, an agonist antibody to FAS, TRAILreceptor, or TNFR. In addition, aptamers and other synthetic ligandscapable to activate the corresponding receptors may be used.

It also is possible to diagnose whether a tumor in a patient is capableof being treated by a present carbazole compound. A sample of the tumoris obtained from the patient. Cells of the tumor then are transducedwith a p53 reporter system, such as a p53-responsive lacZ reporter. Thetransduced cells then are incubated with the compound. The production ofa p53-mediated signal above controls indicates that the tumor can betreated by the carbazole compound.

FIGS. 1A and 1B are plots showing that the present carbazoles inhibitNF-κB transcriptional activity in TNF-treated cells (FIG. 1A) and acomparison of active concentration (EC₅₀ values) on p53 activation andNF-κB inhibition (FIG. 1B).

The present carbazole compounds have significant anti-cancer propertiesin vitro (FIGS. 2A-K) and in vivo (FIG. 3). FIGS. 2A-K show the effectof various carbazole compounds on tumor cells differing in their p53states after treatment for 1 hour with different concentrations of fourpresent carbazole compounds. Cell survival was assessed at 72 hours bymethylene blue staining. Therefore, it is theorized, but not reliedupon, that p53 activation by the present carbazole compounds may not bethe primary death-inducing signal, but may rather reflect a type of cellstress caused by inactivation of constitutively active NF-κB.

The tumor cells tested were HCT116 colon adenocarcinoma p53 wt (FIG.2A), MDA-MB-231 breast adenocarcinoma p53 mut (FIG. 2B), DLD1 coloncarcinoma p53 wt (FIG. 2C), A549 lung adenocarcinoma p53 wt (FIG. 2D),Caki1 renal cell carcinoma p53 wt (FIG. 2E), HT29 colon adenocarcinomap53 mut (FIG. 2F), H1299 lung adenocarcinoma p53 deletion (FIG. 2G),MCF7 breast adenocarcinoma p53 wt (FIG. 2H), RCC45 renal cell carcinomap53 wt (FIG. 2I), ACHN renal cell carcinoma (FIG. 2J), and HT1080 lungfibrosarcoma (FIG. 2K). The tumor cell tested in FIG. 3 is the HCT116 scxenograft model.

The present carbazole compounds analyzed did not induce DNA damage(Table I). It therefore is theorized, that the cytotoxicity of thepresent carbazole compounds results from a unique type of non-genotoxiccell stress involving NF-κB suppression to which cancer cells are moresensitive than normal cells. This illustrates that the presentcarbazoles are a highly effective, novel class of anti-cancertherapeutics.

TABLE I Summary of the effects of present carbazole compounds anddoxorubicin to compare DNA and DNA-damage responsive signaling. Topo IIinhibition cellular γH2AX ATM/ATR Chk1/2 p53 in in Compound localizationstaining activation phospho phosphor cells vitro Ames test Compound 100cytoplasmic no no no S392 no ligation negative Compound 6h cytoplasmicno no no S392 ND ligation negative Example 3 cytoplasmic no no no ND NDligation negative doxorubicin nuclear yes yes yes S15, 46, 392 yesrelegation negative ND—not determined

In accordance with an important feature of the present invention, athree dimensional (3D) superimposition of conformers of active andinactive carbazole molecules revealed characteristics of the compoundsthat contributes to their activity. One important characteristic is theplanarity of the carbazole core. Comparing multiple three dimensionalalignments of the conformers, it was found that in all active carbazolecompounds, the carbazole core region was planar (FIG. 4). Inactivecompounds can be planar or non-planar (FIG. 5). Additional structuralstudies of compounds having similar atom distributions over themolecular architecture confirmed that planarity of the carbazole ringarea is important in determining potency of p53 activation by thecarbazole compound (FIGS. 6 and 7). An active carbazole, i.e., Example2, is shown in FIG. 6. An inactive carbazole, i.e., Compound 200, isshown in FIG. 7. Compound 200 has a structural formula:

The finding that carbazole compounds able to activate p53 have planarstructure led to an unrelied upon hypothesis that the mechanism ofaction of these compounds is mediated via DNA intercalation. It ishypothesized that the correlation between p53 activation potency and theplanar structure of carbazole core reflects an ability to intercalateDNA. To test this hypothesis, the carbazole compounds were virtuallyintercalated into the three dimensional DNA structure taken from thep53-DNA complex (1TUP PDB structure). The initial intercalation wasperformed as follows. An active carbazole molecule, i.e., Compound 300,was superimposed upon the DNA structure such that the planar ring areawas positioned between and parallel to two stacking base pairs. Theintercalated molecule then was placed against Arg280 of p53. The Arg280residue is considered to be crucial for p53-DNA interaction. See M.Kitayner et al., Molecular Cell, 22, pages 741-753, June 2006. Such abrute force superimposition violates Van der Waals interactions betweenatoms of the two structures. Using the MOLOC molecular mechanicssoftware package, the tertiary DNA-carbazole analog-p53 complex wasoptimized to reduce Van der Waals and torsion angle tensions in thecombined DNA-molecule structure. After the optimization, the position ofthe active molecule was fitted to the cavity in DNA with the ring planethat is parallel to DNA stacking base pairs. As a result of thisoptimization procedure, hydrogen bond acceptors (HBA) located on thecarbazole ring substituents were positioned in the major groove and theside chain attached to the carbazole nitrogen was positioned in thenarrow minor groove. The structural formula for Compound 300 is:

As a result of this study, a dsDNA fragment with an inside cavity bettershaped to accommodate p53 activating carbazole compounds was created.Using the GOLD software package, molecular docking was performed on avariety of carbazole compounds with known p53 activity. It wasdemonstrated that, on average, highly active molecules (p53 activationEC₅₀<130 μM) were much better fitted to this cavity than molecules withEC₅₀>130 μM. The conformers of highly active carbazole compounds areuniformly positioned inside the cavity, whereas the quality of fitdiminished among carbazole compounds having weak p53 activation potency.The majority of inactive molecules were characterized by very poorquality of fit.

A proper positioning of the substituent attached to the carbazolenitrogen into the minor groove of DNA may be important for achievinggood p53 activity. Simply, the fit of the side chain into the minorgroove improves the overall fit of the carbazole compound. In addition,the atom voting analysis of the three dimensional superimposition ofactive carbazole compounds shows that a positively charged amino groupin the side chain is important for achieving activity.

The positions of HBA (hydrogen bonding) atoms on the carbazolesubstituents, and their identity are important to achieve p53 activity.The low quality of HBA atoms (i.e., nitrogen) leads to a weak activityof the carbazole compound. The absence of HBA atoms on carbazolesubstituents renders a compound inactive. All highly active compoundshave non-rotated carbazole substituents. The docking of highly active(EC₅₀<130 nM) and active molecules (EC₅₀ about 1 nM) into the DNA cavitydemonstrates that rotated carbazole substituents with good HBA atoms cancreate hydrogen bonds with DNA atoms. In contrast, the non-rotatedcarbazole substituents do not hydrogen bond with DNA, which leads totheir high activity.

The antitumor activity of Example 7 (Compound 6h) was demonstrated usingthe B16 melanoma singenic tumor model as follows. C57BL/6 mice wereinoculated intradermally at 2 sites of the abdomen with 5×10⁴ murine B16melanoma cells. When at least one of the tumor inoculation sitesdeveloped a tumor (average size about 6 mm³), treatment commenced. Micewere treated daily by oral gavage for up to 14 days with either 0.5%methylcellulose vehicle control or 30 mg/kg Example 7 (n=5mice/treatment group). Tumor measurements were collected by digitalcalipers every 1-2 days. The effect of treatment on individual tumors ispresented in FIGS. 8A and 8B. With the exception of one mouse fromExample 7 treatment group, no effect was observed on overall mouseweight for either treatment group (<10% changes). By Day 9, there was anapproximately 3-fold decrease in tumor growth in Example 7 treatmentgroup compared to the vehicle control group (68% growth suppression).

To confirm the anti-tumor activity of Example 7, the carbazole compound(30 mg/kg) was delivered orally using the HCT116 xenograft model. Inthis test, athymic nude mice were inoculated with 5×10⁶ of HCT116 tumorcells into two sites. 90% of tumors appeared between the seventh andeleventh days after inoculation. Oral daily treatments with 30 mg/kg ofthe compound of Example 7 in 0.5% methylcellulose or a 0.5%methylcellulose vehicle control began when at least one tumor per mousereached 20-25 mm³ in size. The mice were treated until control tumorsreached 1000 mm³. Following commencement of treatment, mice weremonitored for overall condition, weight loss, and survival, as well asfor the size of tumors measured every other day.

TABLE 2 Experimental groups Tumor Number Mode of measurement; Group ofmice Cell line Treatment administration Dose schedule monitoring 1 10HCT116 0.5% po Once a day D1-D5, Every other day (control)methylcellulose D8-D12, D15-D19 once tumors are once tumors reachvisible; daily 20-25 mm³; volume monitoring; of 250 ul per 25 g mouseweights mouse weight every other day 2 10 HCT116 7.5 mg/kg of po Once aday D1-D5, Example 7 in D8-D12, D15-D19 0.5% once tumors reachmethylcellulose 20-25 mm³; volume of 250 ul per 25 g mouse weight D—day

In this test, 100% of the mice in Group 2 survived the experiment. Noweight loss was observed in control vehicle treated group 1, whereas ingroup 2, three mice lost 15-20% weight by the end of experiment. Theweight of the remainder of the mice in group 2 fluctuated in the rangeof 95-105% of original weight. No any other abnormal signs were noticedin mouse appearance, activity, or behavior of group 2.

In control group 1, HCT116 tumors grew exponentially, as expected within a regular deviation between faster and slower growing tumors. InExample 7 treated group 2, growth of all tumors was delayed comparedwith vehicle control treated group 1. On day 14 after start oftreatment, no treated tumors attained the size of the slowest growingcontrol tumor (530 mm³). In average growth of tumors, treatment withExample 7 suppressed tumor growth by a factor of about 3.5 (73% ofinhibition) compared to vehicle control group 1. Importantly, onetreated tumor was completely cured, and, on autopsy, only a minimalconnective tissue type formation was found in place of the tumor. Thesize of several treated tumors not only increased more slowly thancontrol tumors, but decreased, which is indicative of tumor cell deathand tumor destruction as a result of treatment with the compound ofExample 7. This result was observed for the first 3-5 days of treatment,then the treated tumors continued to slowly grow.

This experiment shows that the compound of Example 7 has a suppressiveeffect on the growth of HCT116 subcutaneous xenograft tumors in nudemice. The growth suppression value is 73%, which is considered by NCIstandards as a significant anti-cancer activity (≥42%). The aboveresults are illustrated in FIGS. 3 and 8A-B, 9A-C, and 10A-B.

FIG. 3 is an average curve of tumor growth showing treatment of HCT116colon carcinoma xenograft tumor with the compound of Example 7 and acontrol (MC). Tumors treated with the compound of Example 7 exhibit asubstantially reduced tumor volume. The bars in the graph of tumorvolume vs. days of treatment represent the standard deviation. FIGS.8A-B contains plots showing the growth of individual tumors treated inmice with the control vehicle (FIG. 8A) and with the compound of Example7 (FIG. 8B).

FIGS. 9A-C shows the growth of individual tumors, up to 100 mm³, in micetreated with the compound of Example 7. FIGS. 10A-B show that tumor sizedecreased during the first days of treatment, and tumors that werecured.

FIG. 11 demonstrates a carbazole compound, i.e., Compound 100, thatactivates p53 in the above-described assay exhibits a significantanti-cancer activity against a wide variety of tested cancer cells. Eachof the cell lines presented in FIG. 11 was seeded into 96 well plates.The next day, cells were treated with a range of concentrations ofCompound 100. Treatment occurred for 24 hours, at which timecompound-filled medium was replaced with compound-free medium and cellswere allowed to grow until the control wells, which were treated withonly vehicle control (DMSO), reached a monolayer (typically within 48hours). Cells then were fixed and stained with 0.5% methylene blue in50% methanol. The dye was eluted with 1% SDS and the absorbance measuredat 650 nm. Data is presented as the absorbance at 650 nm versus theconcentration of Compound 100.

The compounds of Examples 7, 15, and 13 showed a potent anticanceractivity. In an efficacy test using these three compounds, athymic nudemice were inoculated subcutaneously with a suspension of Caki1 humanrenal cell carcinoma cells into both rear flanks. When at least onetumor in a mouse reached 20-50 mm³, treatment was commenced. Mice weretreated orally by gavage with either 30 mg/kg of Example 7, 5 mg/kg ofExample 15, or 25 mg/kg of Example 13 formulated in 0.2%hydroxymethylcellulose. As a positive control, one group was treatedwith 40 mg/kg Sunitinib, an approved drug for the treatment of renalcell carcinoma. Treatment was given for 4 weeks on a 5 day on/2 day offschedule. After treatment was completed, mice were monitored for anadditional 4 weeks to determine how quickly tumor growth returned tonormal. All three compounds caused significant tumor growth inhibitioncompared to the vehicle control (Day 24 (end of treatment) Example 7 73%inhibition, Example 15 50% inhibition, Example 13 62% inhibition). Thisantitumor effect of Examples 7 and 13 and, to a lesser extent, Example15 was substantially more than the Sunitinib control (42%).Interestingly, cessation of treatment with any of the present compoundsdid not lead to rapid regrowth of tumor. In contrast, the end ofSunitinib treatment resulted in immediate tumor growth such that by Day40, there was no significant difference in tumor volume observed betweenthe Sunitinib and vehicle control groups.

In summary, all three carbazoles caused tumor growth inhibition thatpersisted even after treatment was completed. The order of potency isExample 7 is greater than or equal to Example 13, which is greater thanExample 15. Treatment with Example 7 caused no apparent side effectscompared to Examples 13 and 15. The severest side effects were observedwith Example 13, where two mice had to be taken off treatment short-termand one mouse was removed permanently due to weight loss that led topremature euthanasia. Thus, Example 7 appeared to be the “safest”carbazole in this test. Because Example 15 only caused 10-15% weightloss that was short-term, this compound was the second safest in thistumor model. Based on the results, Example 7 is a preferred compoundwith potent antitumor activity (70-80% inhibition) in the absence ofside effects.

In another test, the antitumor activity and toxicity of Examples 7, 15,and 13, at the maximum tolerated dose (MTD) in nude mice bearing humanHCT-8 ileocecal adenocarcinoma xenografts, and in nude mice bearinghuman HT-29 colon adenocarcinoma xenografts, were evaluated. Acomparison of the antitumor efficacy and toxicity of Examples 7, 15, and13 at the MTD side by side against human HCT-8 and HT-29 colonxenografts also was performed.

As shown above, Examples 7, 15, and 13 demonstrated antitumor efficacyagainst various human tumour xenografts sc in nude or SCID mice (such asHCT-16, DLD1, Caki1 and MDA-MB-231 tumors, with the tumor cellsinoculated as a cell suspension in to the mice). The in vivo antitumorefficacy and toxicity of these three compounds were evaluated at theirrepetitive MTD against HCT-8 (relatively sensitive to chemotherapy, suchas 5-FU and irinotecan) and HT-29 (relatively resistant to chemotherapy)xenografts established in nude mice by transplanting about 50 mg tumorpieces. In this study, the antitumor effect and toxicity of the threecarbazoles against HCT-8 and HT29 colon cancer tumors were compared withtreatment beginning when the tumor size reached 150-200 mg (about 7 daysafter tumor transplantation).

Materials and Methods

Animals. Eight to twelve-week-old female athymic nude mice (nu/nu, bodyweight 22-25 g) were obtained from Harlan Sprague Dawley Inc.(Indianapolis, Ind.) and maintained at five mice/cage with water andfood ad libitum according to an institutionally approved animalprotocol.

Drugs. All three compounds were formulated in 0.2%hydroxypropylmethylcellulose at concentrations of 0.5 mg/ml for Example15, 3 mg/ml for Example 7, and 2.5 mg/ml for Example 13.

Tumors. Human ileocecal adenocarcinoma HCT-8 and colon adenocarcinomaHT-29 xenografts were used. The xenografts were initially established byinjecting s.c 10⁶ cultured cells and tumors were passed severalgenerations by transplanting about 50 mg non-necrotic tumor (2-3 pieces)via a trocar from the passage tumors when the tumors reach to 1-1.5 g.

Drug doses and schedule. All the compounds were given by oral (p.o.)administration at the MTD with Example 15: 5 mg/kg/day; Example 7: 30mg/kg/day; and Example 13: 25 mg/kg/day, 5 days a week for 4 weeks (5days/week×5) or until the mouse had to be sacrificed due to a largetumor. Treatment was initiated 7 days after tumor transplantation whenthe tumors reached 150-200 mg. The mice in the control group receivedthe vehicle (0.2% hydroxypropylmethylcellulose) at 200 μl per 20 g mousebody weight (same as the treatment groups). Five mice were used for eachexperimental group with 10 tumors (1 tumor each in the left and rightside flanks).

Tumor Measurement. Two axes (mm) of tumor (L, longest axis; W, shortestaxis) were measured with the aid of a Vernier caliper. Tumor weight (mg)was estimated using a formula: tumor weight=½ (L×W2). Tumor measurementswere taken daily at the same time as drug treatment and 3-4 times a weekof post therapy.

Maximum Tolerated Dose (MTD) and toxicity evaluation. The MTD wasdefined as the highest drug dose that did not cause drug-relatedlethality in mice with a weight loss less than 20% of original bodyweight and toxicities were reversible. The kinetics of drug-inducedtoxicities (body weight loss, diarrhea, and lethality) were determineddaily for the first 10 days after treatment and every two daysthereafter.

Antitumor Activity. Antitumor activity was assessed by maximum tumorgrowth inhibition (MTRI) which is the mean tumor weight of treated group(MTWTG) compared with untreated control group (MTWCG) at the same timepoint (MTRI=MTWTG−MTWCG)÷MTWCG×100%). The tumor doubling time (TDT) wasdefined as the mean time for the tumor to reach twice its initialweight. Tumor response was expressed as partial tumor response (PR) whentumor weight was reduced at least 50% of the initial tumor size andcomplete tumor response (CR) was defined as the inability to detecttumor upon palpation at the initial site of tumor appearance.

Results

(a) The antitumor activity and toxicity of Example 15, 7, and 13 in nudemice bearing HCT-8 xenografts.

The data in the following table show the antitumor activity and toxicityof vehicle control, Example 15, Example 7, and Example 13 administeredp.o. 5 days on and 2 days off per week to nude mice bearing HCT-8xenografts. The data indicate that Example 13 and 15 had moderateantitumor activity against HCT-8 xenografts with inhibitory rates of35-40% and delayed tumor growth by 17% (Example 13) and 57% (Example15), respectively, compared to the vehicle control (double time: 4.8days). The planned four courses of treatment for Examples 13 and 15 werenot completed because of the large tumor volume that required the miceto be sacrificed. Example 7 was much more active than Examples 13 and 15against HCT-8 xenografts with an inhibitory rate of 65.9% and a 142%delay of tumor growth. Example 7 did not produce any PR or CR. Withrespect to toxicity, the vehicle produced mild toxicity with the animalgroup of individual HCT-8 tumor 5 days on and 2 days off a week×2-4weeks.

TABLE Antitumor activity and toxicity of Examples 15, 7, and 13 in nudemice bearing human HCT-8 ileocecal adenocarcinoma xenografts TreatmentAntitumor Activity Toxicity (%) (mg/kg/d) MTGI (%) TDT (day) PR (%) CR(%) MWL lethality Control-Vehicle — 4.8 ± 0.3 0 0  5.5 ± 1.9 0 Example15 (5) 40.0 ± 18.6 7.5 ± 3.5 0 0 10.0 ± 3.8 0 Example 7 (30) 65.9 ± 11.011.6 ± 2.5  0 0 12.2 ± 6.8 0 Example 13 (25) 35.7 ± 20.0 5.6 ± 1.2 0 0 8.5 ± 2.9 0 MTRI: maximum tumor growth inhibition; TDT: tumor doublingtime; PR: partial tumor response; CR: complete tumor response; MWL:maximum weight loss of pretreatment body weight. Control group was given0.2% hydroxypropylmethylcellulose (vehicle)) at 200 μl per 20 g mousebody weight. Five mice were used for each experimental group with 10tumors (in left and right side flanks). (b) The antitumor activity andtoxicity of Examples 15, 7, and 13 in nude mice bearing HT-29xenografts.

The data in the following table show the antitumor activity and toxicityof vehicle control, Example 15, Example 7, and Example 13 administeredp.o. 5 days on and 2 days off per week to nude mice bearing HT-29xenografts, which is more resistant to most chemotherapeutic agentscompared to HCT-8. The data indicate that all three compounds were moreactive against this tumor than HCT-8. Similarly, Example 13 was theleast active compound among the three, with inhibitory rates of 48% anddelayed tumor growth by 50% (the tumor double time for the control was7.8 days). While Example 15 produced similar tumor growth inhibition(58%), it had less of an effect on tumor growth delay (delaying 76% vs122%) compared to Example 7. No PR or CR was produced by the threeagents. With respect to toxicity, Example 7 and 13 produced less bodyweight loss. Example 7 was more toxic in the HT-29 experiment comparedto the HCT-8 experiment with 18% weight loss and 40% lethality.

TABLE Antitumor activity and toxicity of Examples 15, 7, and 13 in nudemice bearing human HT-29 colon adenocarcinoma xenografts. TreatmentAntitumor Activity Toxicity (%) (mg/kg/d) MTGI (%) TDT (day) PR (%) CR(%) MWL lethality Control-Vehicle —  7.8 ± 0.8 0 0 5.6 ± 4.2 0 Example15 (5) 58.4 ± 6.4 13.1 ± 2.3 0 0 17.9 ± 6.8  40 Example 7 (30)  58.5 ±17.4 17.3 ± 4.8 0 0 7.6 ± 5.1 0 Example 13 (25) 47.6 ± 6.7 11.7 ± 1.2  00 4.4 ± 2.2 0 MTRI: maximum tumor growth inhibition; TDT: tumor doublingtime; PR: partial tumor response; CR: complete tumor response; MWL:maximum weight loss of pretreatment body weight. Control group was given0.2% hydroxypropylmethylcellulose (vehicle)) at 200 μl per 20 g mousebody weight. Five mice were used for each experimental group with 10tumors (in left and right side flanks).

CONCLUSION

In conclusion, Examples 15 and 13 show moderate antitumor activity,while Example 7 had better antitumor efficacy against both HCT-8 andHT-29 xenografts;

Example 15 was more toxic than Examples 7 and 15 in the HT-29 study;

Unlike the response to most other chemotherapeutic agents, HT-29xenografts were more sensitive to all three carbazoles compared to theresponse observed with HCT-8 xenografts;

The data shows that Example 7 is a preferred compound against both HCT-8and HT-29 xenografts.

In another experiment, the anti-tumor activity of Example 7 in a murinemodel of neuroblastoma was demonstrated. The N-myc (TH-MYCN) transgenicmice carry the human N-myc oncogene under the control of a tyrosinehydroxylase promoter, which is expressed in neuroectodermal cells duringearly development, and the mice develop a murine equivalent of humanneuroblastoma. These mice have proved to be an excellent model sharingseveral important features with the human disease, including site of thetumor and its metastases, the histology of the tumor, positive stainingfor neuroblastoma-associated marker proteins, the presence of synapsesand neurosecretory granules, gains and losses of chromosomes in regionssynthetic with those observed in human neuroblastoma, and theamplification of copy number of N-myc specifically in the tumors whichdevelop.

Modern chemotherapy has dramatically improved the survival rates formany cancers. However, the development of multi-drug resistance in theclinical setting for neuroblastoma is one of the major causes oftreatment failure, and circumventing multi-drug resistance has enormousclinical potential. Drugs that target novel pathways not previouslyimplicated in neuroblastoma could provide a new avenue for treatmentprotocols.

The test results showed that the compound of Example 7 has a remarkableanti-tumor activity in this model of neuroblastoma, although high dosesproved toxic in some mice. Mice treated with 30 mg/kg of Example 7 lostweight rapidly and unexpectedly (2 g overnight in some case) and werefound dead with small spleens and signs of dehydration. Mice thatsurvived therapy were tumor free for a period extending from 15 to 47days, however all mice were not treated according to the same schedule.Doses were altered according to weight loss. Based on the results,Example 7 exhibits good efficacy in the TH-MYCN model of neuroblastoma.

Modern chemotherapy has dramatically improved the survival rates formany cancers. However, the development of multi-drug resistance in theclinical setting for neuroblastoma is one of the major causes oftreatment failure, and circumventing multi-drug resistance has enormousclinical potential. Drugs that target novel pathways not previouslyimplicated in neuroblastoma could provide a new avenue for treatmentprotocols.

Daily administration of Example 7 also prevents tumor onset in anMMTV-neu transgenic mouse model of mammary cancerogenesis.

Breast cancer (BC) is a serious health care issue due to high incidenceof this malignancy and limited success of available treatments. It isknown that the family history of this disease along with severalspecific genetic factors with substantial degree of probabilitypredispose individuals to BC. Therefore, women from high BC risk groupstheoretically can benefit from a preventive anti-BC therapy. The presentcarbazole compounds can modulate several cancer-related cellularpathways in a direction that leads to tumor growth suppression. Thecompound of Example 7 causes no serious side effects in mice whenadministered at therapeutic doses (20-25 mg/kg). Example 7 showed nomutagenic activity in Ames assay. In this study, Example 7 was tested asa preventive agent for BC in mice.

Animal model: MMTV-neu female mice on the background of FVB strainexpress non-activated Her2 proto-oncogene under MMTV promoter responsiveto estrogen stimulation. The mice develop spontaneous mammary tumorsstarting from 24 weeks of age with 70-80% of animals normally developingtumors by 10 months of age. The objectives of this test were to (a)compare tumor incidence in mice treated with Example 7 vs. vehiclecontrol (water), and (b) compare weight gain and general appearance ofmice treated with Example 7 vs. vehicle control.

Study design: 40 female mice were weaned from breeding parents at 21days of age and placed in separate cages (4 mice/cage). At this moment,mice were labeled and assigned to treatment or control groups (20 micein each). Body weight in both groups was measured once a week. Startingfrom 4 weeks of age, mice from the treatment group were provided withwater containing Example 7. Liquid consumption in treatment and controlgroups was estimated every day. Based on these measurements, liquidconsumption per gram of mouse weight was calculated, and Example 7concentration in drinking water was adjusted to desirable therapeuticdose. Fresh solutions of Example 7 were prepared weekly. Mice weremonitored once a week for tumor formation by the palpation of mammaryglands. Mice were sacrificed at a moment when cumulative tumor sizereached the volume of 1000 mm³.

Number Group of mice Targeted dose Delivery mode 1 20 None (water) Dailydrinking 2 20 about 25 mg/kg Example 7 Daily drinkingPreliminary results: Mice from the treatment group were consumingExample 7 at average rate of 20 mg/kg daily. Variability is due tolimited accuracy of drug delivery via drinking water. No differencesbetween treatment and control group were observed in weight distributionor abnormalities in animal appearance.

In the course of the experiment several spontaneous deaths occurred inthe treatment and control groups. The cause of death was not clearbecause the animals had no tumors and post-mortem gross pathologyexamination showed no obvious abnormalities. None of the mice diedearlier than several weeks after the beginning of Example 7administration. During the test, three control mice died at an age ofabout 15, 41 and 42 weeks, respectively. Seven mice died in the Example7 group at different ages ranging from 12 to 45 weeks (from 7 to 41weeks since the beginning of the experimental treatment).

Twenty-one and 19 mice reached tumor bearing age in the control andtreatment groups, respectively. Among them, a tumor was developed in 14(67%) of control mice and 7 (37%) of mice receiving Example 7.

This test showed:

(a) chronic administration of Example 7 with drinking water at about 20mg/kg per day caused no changes in mouse body weight;

(b) a higher death rate in Example 7 treated group versus control group,but the difference was not statistically significant and there was nocorrelation between length and treatment period and death of mice; and

(c) a lower rate of tumor burden among mice treated with Example 7versus control animals.

Carbazole compounds of the present invention also exhibit antiparasiteactivity. In particular, the effect of carbazole compounds on themalaria parasite was tested by culturing Plasmodium falciparum: (strainD10) in vitro in the presence or absence of test compounds. Active andinactive carbazole compounds (with respect to activating p53) wereselected for tested, as was quinacrine, a conventional anti-malariaagent. Table 3 summarizes the structures of the compounds, together withtheir EC₅₀ values in the p53 activation assay.

TABLE 3 Test compounds Compound Structure EC₅₀, μM Example 2

0.64 Compound 400

Inactive Example 7

0.37 Compound 500

Inactive Example 18

0.09 Quinacrine (QC)

about 5

Example 2, Compound 400, and Example 7 were tested at 29, 57, and 143 nMconcentrations. Compound 500, Example 18, and quinacrine were tested at143 nM only.

In each experiment, 1000 erythrocytes were assessed microscopically todetermine the number of infected cells. In control experiments (no testcompounds added or PBS added), the percentage of infected cells variedbetween 1.6% and 6.4%. Infectivity reduction indexes determined in thestudy are shown in FIG. 12. Potency of p53 activation assay clearly, butindirectly, correlated with the inhibition of Plasmodium falciparum: invitro. Three compounds active in the p53 activation assay (Examples 2,7, and 18) demonstrated anti-malarial activity comparable to quinacrine.Compounds inactive in the p53 activation assay (Compounds 400 and 500)showed no parasitemia reduction.

The compounds of Examples 7, 13, and 15 also showed anti-protozoanactivity. Human African trypanosomiasis (HAT) or “sleeping sickness” isone of the most important, but equally most neglected, tropicalinfections. It is caused by a protozoan, Trypanosoma brucei, which istransmitted to humans through the bite of a tsetse fly (Glossina spp).Although nearly eliminated in the 1960s, HAT has reappeared on anepidemic scale in a number of sub-Saharan areas inhabited by the tsetsefly. According to the World Health Organization, about 500,000 peoplecurrently carry trypanosomes and will die if left untreated. The highmortality associated with HAT is due, at least in part, to a lack ofefficacious drugs that can be easily administered.

The drugs currently used to treat HAT are old, toxic, and difficult toadminister in the field. Suramin and pentamidine, which are used totreat early stage disease, must be injected in a clinic. However, accessto health clinics is uncommon in the rural areas of Africa where thedisease is endemic. Moreover, many early stage patients do not seektreatment because they are unaware that they are infected. This is dueto poor screening of at risk populations, as well as the variable,intermittent, and relatively mild nature of early stage HAT symptoms. Bythe time obvious symptoms emerge, patients are often already in the latestage of the disease. Treatment of late-stage HAT is especiallytroublesome because it involves injection of melarsoprol, an arsenicalthat literally burns patients on injection. This treatment is so painfulthat many patients refuse treatment and have to be physically restrainedand forced to receive the medication. Moreover, melarsoprol hassignificant adverse side effects that result in the death of 5-20% oftreated patients. For these reasons, as well as the expected problem ofdrug resistance, there is an urgent need to create a pipeline of new,orally bioavailable drugs to take the place of the current antiquatedmedications.

Tests have shown that the compounds of Examples 7, 13, and 15 haveremarkable anti-T. brucei activity (low nanomolar IC₅₀) in vitro.Preliminary T. brucei inactivation experiments were performed in vitro.The life cycle of T. brucei involves a “procyclic” developmental stagein a tsetse fly, and a “bloodstream” form in humans. All studies wereperformed with the bloodstream stage of the parasite, because it is thedisease-causing developmental stage.

To evaluate the relative anti-trypanosome activities of Examples 7, 13,and 15, and to determine the concentration of each compound that killed50% of the parasites (IC₅₀), T. brucei was exposed to a range ofcompound concentrations. Suramin, a conventional anti-HAT drug, was usedas a positive control.

Bloodstream T. brucei was seeded (3×10³ cells/mL) in 24-well (500 uL ofthe media per well) plate. The concentrations of the compounds (0-200 nMfor Example 7, 0-20 nM for Example 13, and 0-3 nM for Example 15) wereadded to the cells (duplicate cultures). Control cultures received equalvolumes of DMSO. All three test compounds completely eliminated T.brucei because no parasites were detected after 48 hrs of culture in thepresence of the drugs. Control cultures treated with DMSO (vehicle)contained parasites at a density of 3×10⁶/mL.

Trypanocidal activity increased in the order Example 7 less than Example13, which is less than Example 15, with IC₅₀ values of about 43 nM,about 6-11 nM, and about 0.5 nM, respectively. The IC₅₀ of Suramin isgreater than 300 nM.

Therefore, compounds of the present invention are excellent fordevelopment as trypanocidal drugs.

The compound of Example 15 was tested for activity against differentfungi strains. Example 15 in vitro susceptibility testing was done bythe CLSI M27A3 and M38A2 methods for 31 clinical and laboratory fungalstrains. In general, the strains selected demonstrate differentsusceptibility patterns for current antifungal drugs. For the evaluationof Example 15, 8 Aspergillus spp., 21 Candida spp., 1 Cryptococcusneoformans, and 1 Debaryomyces hansenii strains were tested.

Aspergillus Spp. Strains:

Eight Aspergillus spp. strains were used throughout the study. Six A.fumigatus, one A. flavus and one A. terreus also were used. The A.fumigatus group included two itraconazole-resistant strains (RIT13 andRIT5 strains) (1), one triazole cross-resistant strain (MUT10) (2, 3),one echinocandin cross-resistant strain (EMFRS678P) (4), and 2 wild typesusceptible isolates (R21 and ATCC13073). The “non-fumigatus” strainswere susceptible to all available antifungal with the exception of theA. terreus isolate (naturally less susceptible to amphotericin B) (5).

Candida spp. strains: 20 Candida spp. clinical isolates and onelaboratory control strain (C. albicans SC5314) was used in the study. Inthe collection were included:

5 C. albicans isolates, two wild type strain (SC5314 and ATCC 36082),one echinocandin resistant isolate (M205) (6), and two fluconazoleresistant clinical isolates (3795 and 3184) (7).

3 C. krusei strains, 1 ATCC control strain (ATCC 6258 CLSI control), and2 isogenic clinical strains (98 and 100). 100 is echinocandin crossresistant (8, 9)

2 C. glabrata strains, one wild type clinical strain (3168), and oneechinocandin cross-resistant isolate (3830) (10).

2 C. tropicalis strains, one ATCC control strain (ATCC 750), and oneechinocandin cross-resistant (T3) (11).

2 C. dubliniensis strains one wild type strain (3949) and the other withechinocandin paradoxical effect (M204).

4 naturally reduced echinocandin susceptibility Candida spp. (12): 1 C.orthopsilosis (981224), 1 C. metapsilosis (2006-113), C. parapsilosis(ATCC 22019 CLSI control), and C. guilliermondii (ATCC6260) (13).

Other Candida spp. and genera: 1 C. rugosa (M83), 1 C. lipolytica(M159), C. lusitaniae (200450), 1 Cryptococcus neoformans (499), and 1Debaryomyces hansenii.

Susceptibility Testing:

Example 15 in vitro susceptibility testing was done using the CLSIstandardized methods for yeast (M27A3) (14) and for molds (M38A2) (15).The MIC (minimum inhibitory concentration) readings were performed at 24and 48 hours.

TABLE 1 In vitro susceptibilities of fungal strains against Example 15Example 15 MIC* MIC* Strain Organism Genotype Phenotype (24 hs) (48 hs)ATCC13073 A. fumigatus WT Susceptible 2.56 5.13 R21 A. fumigatus WTSusceptible 1.28 2.56 MUT10 A. fumigatus Cyp51ApG138C Triazole crossresistant 0.66 1.28 RIT5 ^(a) A. fumigatus AfuMDR3 and AfuMDR4Itraconazole resistant 0.66 1.28 constitutively overexpressed RIT13 ^(a)A. fumigatus AfuMDR3 and AfuMDR4 Itraconazole resistant 1.61 1.28overexpression induced by azoles plus Cyp51Ap G54E EMFRS678P A.fumigatus Fks1pS678P Echinocandin cross- 1.28 2.56 resistant AFCLF9128A. flavus WT Susceptible 0.33 0.33 At123 A. terreus WT Intrinsically AMB0.08 1.28 resistant SC5314 C. albicans WT Susceptible 0.04 0.08ATCC36082 C. albicans WT Susceptible 0.04 0.04 M205 C. albicansFks1pS645P Echinocandin cross- 0.16 0.33 resistant 3795 C. albicansErgl1pF136L/K134R Fluconazole-resistant 0.16 0.16 3184 C. albicans CDR1overexpression Fluconazole-resistant 0.33 0.66 3168 C. glabrata WTSusceptible 0.16 0.16 3830 C. glabrata Fks2pS663P Echinocandin cross-0.16 0.16 resistant ATCC 6258 C. krusei WT Susceptible (naturally 0.330.66 fluconazole resistant) 98^(b) C. krusei WT Susceptible (naturally0.33 0.33 fluconazole resistant) 100^(b) C. krusei Fks1pF655F/CEchinocandin cross- 0.33 0.33 resistant ATCC750 C. tropicalis WTSusceptible 0.08 0.16 T3 C. tropicalis Fks1pS645S/P ^(a) Echinocandincross- 0.04 0.08 resistant 3949 C. dubliniensis WT Susceptible 0.16 0.16204 C. dubliniensis WT Echinocandin 0.16 0.16 paradoxical effect M159 C.lipolytica WT Susceptible 0.02 0.08 M83 C. rugosa WT Susceptible 0.160.66 200450 C. lusitaniae WT Susceptible 0.04 0.16 ATCC6260 C.guilliermondii WT Echinocandin cross- 0.08 0.16 reduced susceptibilityATCC 22019 C. parapsilosis WT Echinocandin cross- 0.08 0.08 reducedsusceptibility 981224 C. orthopsilosis WT Echinocandin cross- 0.16 0.16reduced susceptibility 2006-113 C. metapsilosis WT Echinocandin cross-0.16 0.16 reduced susceptibility 20124 Debaryomyces WT Susceptible 0.160.16 hansenii 499 Cryptococcus WT Susceptible 0.02 0.08 neoformans*Geometric mean of two repetitions in μM. ^(a) Amino acid numbercorresponding to the C. albicans equivalent.

Aspergillus spp. Example 15 susceptibilities: The A. flavus and A.terreus strains are more sensitive to Example 15 than A. fumigatusstrains (24 hs MIC Geom. Means 0.40 and 1.59 μM, respectively).Moreover, there were no MIC differences between susceptible and azole orechinocandin resistant strains (Table 1).

Yeast Example 15 susceptibilities: There were no Example 15 MICdifferences between azole- or echinocandin-resistant or -susceptibleisolates. On average, yeast MICs were 8-fold lower than Aspergillus spp.MIC (Table 1). MICs obtained for Example 15 are compatible or betterthan MICs for conventional antifungal drugs. Azole and echinocandinresistant strains are sensitive for Example 15.

Effect of Example 15 on Aspergillus fumigatus viability: The antifungalactivity of Example 15 on the Af293 strain of A. fumigatus using the XTTviability assay were evaluated. This strain was selected because it isone of the best characterized strains and was the strain used forsequencing the A. fumigatus genome. XTT viability assay is based on thereduction of the tetrazolium salt (XTT) in the presence of menadione asan electron-coupling agent. There is a relationship between the numberof viable fungi and the amount of XTT reduction (16).

Methods

The same methods previously described in performing the XTT assay (17)were used. Af293 conidia were plated on Sabouraud BHI slants withchloramphenicol and gentamicin (Becton Dickinson, Md.), incubated for 7to 10 days at room temperature, and harvested by washing the slant with10 ml of 0.05% Tween 20 in normal saline (NS). The conidial suspensionthen was passed through a 100-μm filter, counted on a hemacytometer, anddiluted to 2.0×10⁶ CFU/ml. Conidia then were diluted 1:50 in MOPS(morpholinepropanesulfonic acid)-buffered RPMI (pH 7.0). A stocksolution of Example 15 dissolved in DMSO was used. Example 15 wasdiluted in MOPS-buffered RPMI (pH 7.0) (final concentration of DMSO: 2%prior to addition to fungal suspensions). Following the addition ofdrug-containing medium to each well, the conidial suspension (100 μl)was added (t=0 h). Control wells contained conidia, medium, and vehiclewithout drug. Blank wells consisted of medium only without conidia. Aninitial experiment using a wide range of concentrations of Example 15without added fungus showed that the Example 15 reagent did not affectoptical density in the XTT assay. Plates were incubated at 37° C., andthe XTT assay was performed at 24 h essentially as previously described(16), but with a minor adaptation. A stock solution of XTT (SigmaChemical, St. Louis, Mo.) was dissolved in NS (1 mg/ml). A 10-mMsolution of the electron-coupling agent menadione (Sigma Chemical) wasprepared in acetone and then diluted 1:10 in NS. A working solutionconsisting of 4.0 ml XTT and 0.5 ml menadione was prepared immediatelybefore use. Fifty microliters of the combination solution was added toeach well, and plates were incubated for 2 h at 37° C. One hundredmicroliters of the supernatant was transferred to a new plate, and theoptical density at 450 nm (OD₄₅₀) was determined by using a LabsystemsMultiskan Plus plate reader. Final concentrations of XTT and menadionein each well were 200 μg/ml and 25 μM, respectively.

Example 15 had antifungal activity against Af293. The IC50 wasapproximately 2.5 μM. Complete suppression of fungal growth occurred atExample 15 concentrations greater than 12.5 μM, based on visualinspection of the wells.

-   1. A. M. Nascimento, et al., 2003, 47:1719-26.-   2. G. Garcia-Effron, et al., 2008, J Clin Microbiol, 46:1200-6.-   3. S. J. Howard, et al., 2006, Int J Antimicrob Agents, 28:450-3.-   4. E. M. Rocha, et al., 2007, Antimicrob Agents Chemother,    51:4174-6.-   5. W. J. Steinbach, et al., 2004, Clin Infect Dis., 39: 192-8.-   6. G. S. Garcia-Effron, et al. Antimicrob Agents Chemother.-   7. S. Perea, et al., 2001, Antimicrob Agents Chemother, 45:2676-84.-   8. M. Hakki, et al., 2006, Antimicrob Agents Chemother, 50:2522-4.-   9. J. N. Kahn, et al., 2007, Antimicrob Agents Chemother, 51:1876-8.-   10. J. D. Cleary, et al. 2008, Antimicrob Agents Chemother,    52:2263-5.-   11. G. Garcia-Effron, et al. 2008, Antimicrob Agents Chemother,    52:4181-3.-   12. G. Garcia-Effron, et al. 2008, Antimicrob Agents Chemother,    52:2305-12.-   13. D. S. Perlin, 2007, Drug Resist Update, 10:121-30.-   14. Clinical Laboratory Standard Institute, C.L.S.I., 2008, 3rd ed.,    28 (14).-   15. Clinical Laboratory Standard Institute, C.L.S.I., 2008, 2nd ed.,    28(16).-   16. J. Meletiadis, et al., 2001, J Clin Microbiol, 39:3402-8.-   17. Y. F. Brun et al., 2007, Antimicrob Agents Chemother,    51(5):1804-12.

Carbazole compound of the present invention also exhibit antibacterialactivity. In particular, the effect of carbazole compounds of thepresent invention on gram positive (Bacillus subtilis) and gram negative(DH5-α) bacteria was determined by the following procedure, and issummarized in FIGS. 13A and 13B.

In vitro test to evaluate the effect of carbazole compounds on gram(−)and gram(+) bacteria.

Equipment

96 well plate reader (e.g Multiscan, LabSystems, Inc)

Multichannel pipette 50-300 uL range

Filter tips

96 well plates (Corning Costar Cat. No.: 3598)

100 mm petri dishes

Sterile 1 μl inoculation loops (Fisher 22-170-209)

14 ml snap-cap round bottom tubes (Falcon 352059)

Materials

DH5-α (E. coli-gram(−) bacteria)

Bacillus subtilis (gram(+) bacteria)

Difco LB Agar (BD 244510—for DH5-α)

Difco LB Broth (BD 244610—for DH5-α)

Difco Nutrient Broth (BD 233000—for B. subtilis)

Difco Nutrient Agar (BD 212000—for B. subtilis)

Method

Preparation of Reagents

To prepare plates for growing DH5-α, combine 40 g Difco LB agar per 1liter MilliQ water. Autoclave on liquid setting. When the vesselcontaining the solution is cool, pour into 100 mm Petri dishes and leavelids slightly off to prevent build up of moisture as agar gels. Popbubbles that form prior to the agar setting. Store at 4° C.

To prepare medium for growing DH5-α, combine 25 g Difco LB broth per 1liter MilliQ water. Autoclave on liquid setting to sterilize.

To prepare plates for growing B. subtilis, combine 23 g Difco nutrientagar per 1 liter MilliQ water. Autoclave on liquid setting. When vesselcontaining the solution is cool, pour into 100 mm Petri dishes and leavelids slightly off to prevent build up of moisture as agar gels. Popbubbles that form prior to the agar setting. Store at 4° C.

To prepare medium for growing B. subtilis, combine 8 g Difco nutrientbroth per 1 liter MilliQ water. Autoclave on liquid setting tosterilize.

Preparation of Bacteria

Two days prior to the start of the cytotoxicity assay, inoculate DH5-αand B. subtilis into 1 ml of corresponding media from glycerol stocks.Allow to grow during the course of the day at 37° C. with shaking.

Streak each bacteria on the appropriate agar plates using a sterileinoculation loop and allow to grow overnight at 37° C. in the bacteriaincubator.

Using a sterile inoculation loop, pick a single colony of bacteria andinoculate 5 ml of the corresponding media in a 14 ml snap cap tube. Growovernight (˜16 h) at 37° C. with shaking.

The next day, measure the OD600 nm for the culture to ensure thatbacteria is in exponential growth (OD600 nm=0.4-0.8)

Cytotoxicity Assay (1)

Dilute 5 ml cultures of DH5-α and B. subtilis to an OD600 nm of 0.002,which corresponds to about 2×10⁶ cells/ml in a volume sufficient for theexperiment (dependent on the number of plates). Plate out 50 μl intoeach well of X 96-well plates (X=# plates needed for a givenexperiment).

Compounds will be added to plates in 3 fold dilutions from 0.005-50 μMaccording to Table 2.

Chemicals for testing are prepared by dilution of stock solutions in LBBroth or Nutrient Broth for DH5-α and B. subtilis studies, respectively.Cells are treated with the final chemical concentrations presented inthe scheme below (Table 2). Stock solutions are made up in DMSO.Typically, chemicals are made up as 20 mM stock solutions. However, thisconcentration is dependent on the solubility of a given chemical andactual stock concentrations must be noted at time of experiment.

TABLE 4 Scheme of an experimental plate with final concentrations ofchemicals Carbazole compound (Cpd W, X, Y, Z) (μM) Pos Control Neg.Control

Cpd W 100 μg/ml DMSO 0.00762 0.0229 0.0686 0.206 0.617 1.25 1.85 5.5616.67 50 ampicillin Cpd W 100 μg/ml DMSO 0.00762 0.0229 0.0686 0.2060.617 1.25 1.85 5.56 16.67 50 ampicillin Cpd X 100 μg/ml DMSO 0.007620.0229 0.0686 0.206 0.617 1.25 1.85 5.56 16.67 50 ampicillin Cpd X 100μg/ml DMSO 0.00762 0.0229 0.0686 0.206 0.617 1.25 1.85 5.56 16.67 50ampicillin Cpd Y 100 μg/ml DMSO 0.00762 0.0229 0.0686 0.206 0.617 1.251.85 5.56 16.67 50 ampicillin Cpd Y 100 μg/ml DMSO 0.00762 0.0229 0.06860.206 0.617 1.25 1.85 5.56 16.67 50 ampicillin Cpd Z 100 μg/ml DMSO0.00762 0.0229 0.0686 0.206 0.617 1.25 1.85 5.56 16.67 50 ampicillin CpdZ 100 μg/ml DMSO 0.00762 0.0229 0.0686 0.206 0.617 1.25 1.85 5.56 16.6750 ampicillin

Each library chemical to be tested requires 2 rows of a 96-well plate,thus 4 chemicals (e.g. W, X, Y, Z) can be tested in one platesimultaneously. In addition to test chemicals, each plate should includepositive and negative controls. As positive control, ampicillin is usedat 100 μg/ml. As a negative control, DMSO is used in an amount equal tothe highest volume of test compound used (˜0.25% DMSO for 50 μM).

Dilutions of chemicals to be added to the plate are made up as 2×concentrations in appropriate bacteria media and added to correspondingwells in a volume of 50 μl.

Chemicals are diluted in appropriate bacteria media by three-fold serialdilutions starting from 50 μM (e.g. 2× of highest concentration (e.g. 50μM)=100 μM). 300 μl of highest 2× concentration is needed in order touse 1/3 to make 1st dilution and 200 μl media for all subsequentdilutions. 100 μl of the highest 2× concentration is mixed with 200 μlmedia and then 100 μl of the first dilution is mixed with 200 μl ofmedia to produce the next dilution. This process is repeated until all10 2× doses are prepared.

Besides a positive control added in one concentration on each plate, thepositive control of ampicillin is added to each assay run in a fullconcentration range (3 fold dilutions) to ensure proper and robust assayperformance (estimated by comparison of dose response curves amongruns).

Following the addition of carbazole compounds to 96-well platescontaining bacteria, plates are transferred to the 37° C. bacteriaincubator for 48 hours. After the incubation, the OD600 mm for each wellof the 96-well plates will be measured.

Data Analysis

The average OD600 nm of the test compound solutions are compared to thatof the DMSO control by dividing the average OD600 nm of the testsolution by that of the DMSO control and multiplying by 100 to producethe % DMSO control (i.e. % cytotoxicity). The % DMSO control is plottedvs compound concentration to generate sigmoid shaped curves. After threeruns are complete, the raw data for all three runs is used for IC₅₀calculations.

The test results showed that the carbazole compounds of Examples 15, 7,4, 12, 13, 17, and 18 and Compound 18c-1 and demonstrated anantibacterial effect against B. subtilis and HB 101 E. coli over a rangeof potencies. Gram (+) bacteria appeared more sensitive to the presentcarbazole compounds. In addition, several of the carbazole compounds,e.g., Examples 15 and 17, were more effective against bacteria than theampicillin control. The present carbazole compounds thereforedemonstrate a definite antibacterial activity.

The invention claimed is:
 1. A method for treating melanoma, comprisingintraarterially administering to a patient in need thereof an effectiveamount of a compound of a structural formula:

wherein R^(a) is selected from the group consisting of C₁₋₆ alkyl, C₁₋₆haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e),N(R^(e))₂, and SR^(e); alternatively, either R^(a) and R¹ or NR^(e) andR¹ together with the carbon atoms to which they are attached form a fiveor six-membered aliphatic carbocyclic or heterocyclic ring; R^(b) isselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e), N(R_(e))₂, andSR^(e), alternatively, either R^(b) and R⁶ or NR^(e) and R⁶ togetherwith the carbon atoms to which they are attached form a five orsix-membered aliphatic carbocyclic ring or a five or six-memberedaliphatic carbocyclic or heterocyclic ring; R^(c) is selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(e), or R^(c) and R^(d)are taken together to form a five, six, or seven-membered aliphaticring, optionally containing an oxygen atom; R^(d) is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, and C(═O)R^(e), or R^(d) and R⁷ together with theatoms to which they are attached form a five or six-membered aliphaticring; R^(e), independently, is selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,or two R^(e) groups taken together with a nitrogen to which they areattached to form a five or six-membered aliphatic ring; R¹, R², R³, R⁴,R⁵, and R⁶, independently, are selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,halo, OR^(e), C(═O)R^(e), C(═O)OR^(e), OC(═O)R^(e), C(═O)N(R^(e))₂,C(═O)NR^(e)SO₂R^(e), N(R^(e))₂, NR^(e)C(═O)R^(e), NR^(e)C(═O)N(R^(e))₂,CN, NO₂, CF₃, OCF₃, SR^(e), SOR^(e), SO₂N(R^(e))₂, and OSO₂CF₃; R⁷ isselected from the group consisting of hydrogen, C₁₋₆alkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl; and n is 1, 2, 3, 4, or 5, or apharmaceutically acceptable salt or hydrate thereof and wherein theintraarterial administration is intraarterial infusion.
 2. The method ofclaim 1, wherein the compound is:


3. The method of claim 1, wherein the melanoma is malignant melanoma. 4.The method of claim 3, wherein the malignant melanoma is metastatic. 5.The method of claim 1, wherein the intraarterial administering occurs ata frequency of one to four times per day.
 6. The method of claim 5,wherein the intraarterial administering occurs at a frequency of onetime per day.
 7. The method of claim 1, wherein the intraarterialadministering occurs at a frequency of one time per week.
 8. A methodfor treating metastatic colon cancer, comprising intraarteriallyadministering to a patient in need thereof an effective amount of acompound of a structural formula:

wherein R^(a) is selected from the group consisting of C₁₋₆ alkyl, C₁₋₆haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e),N(R^(e))₂, and SR^(e); alternatively, either R^(a) and R¹ or NR^(e) andR¹ together with the carbon atoms to which they are attached form a fiveor six-membered aliphatic carbocyclic or heterocyclic ring; R^(b) isselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e), N(R_(e))₂, andSR^(e), alternatively, either R^(b) and R⁶ or NR^(e) and R⁶ togetherwith the carbon atoms to which they are attached form a five orsix-membered aliphatic carbocyclic ring or a five or six-memberedaliphatic carbocyclic or heterocyclic ring; R^(c) is selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(e), or R^(c) and R^(d)are taken together to form a five, six, or seven-membered aliphaticring, optionally containing an oxygen atom; R^(d) is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, and C(═O)R^(e), or R^(d) and R⁷ together with theatoms to which they are attached form a five or six-membered aliphaticring; R^(e), independently, is selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,or two R^(e) groups taken together with a nitrogen to which they areattached to form a five or six-membered aliphatic ring; R¹, R², R³, R⁴,R⁵, and R⁶, independently, are selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,halo, OR^(e), C(═O)R^(e), C(═O)OR^(e), OC(═O)R^(e), C(═O)N(R^(e))₂,C(═O)NR^(e)SO₂R^(e), N(R^(e))₂, NR^(e)C(═O)R^(e), NR^(e)C(═O)N(R^(e))₂,CN, NO₂, CF₃, OCF₃, SR^(e), SOR^(e), SO₂N(R^(e))₂, and OSO₂CF₃; R⁷ isselected from the group consisting of hydrogen, C₁₋₆alkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl; and n is 1, 2, 3, 4, or 5, or apharmaceutically acceptable salt or hydrate thereof and wherein theintraarterial administration is intraarterial infusion.
 9. The method ofclaim 8, wherein the compound is:


10. The method of claim 8, wherein the colon cancer has metastasized tothe liver.
 11. The method of claim 8, wherein the intraarterialadministering occurs at a frequency of one to four times per day. 12.The method of claim 11, wherein the intraarterial administering occursat a frequency of one time per day.
 13. The method of claim 8, whereinthe intraarterial administering occurs at a frequency of one time perweek.
 14. A method for treating metastatic breast cancer, comprisingintraarterially administering to a patient in need thereof an effectiveamount of a compound of a structural formula:

wherein R^(a) is selected from the group consisting of C₁₋₆ alkyl, C₁₋₆haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e),N(R^(e))₂, and SR^(e); alternatively, either R^(a) and R¹ or NR^(e) andR¹ together with the carbon atoms to which they are attached form a fiveor six-membered aliphatic carbocyclic or heterocyclic ring; R^(b) isselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR^(e), N(R_(e))₂, andSR^(e), alternatively, either R^(b) and R⁶ or NR^(e) and R⁶ togetherwith the carbon atoms to which they are attached form a five orsix-membered aliphatic carbocyclic ring or a five or six-memberedaliphatic carbocyclic or heterocyclic ring; R^(c) is selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, and C(═O)R^(e), or R^(c) and R^(d)are taken together to form a five, six, or seven-membered aliphaticring, optionally containing an oxygen atom; R^(d) is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, and C(═O)R^(e), or R^(d) and R⁷ together with theatoms to which they are attached form a five or six-membered aliphaticring; R^(e), independently, is selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,or two R^(e) groups taken together with a nitrogen to which they areattached to form a five or six-membered aliphatic ring; R¹, R², R³, R⁴,R⁵, and R⁶, independently, are selected from the group consisting ofhydrogen, C₁₋₆alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,halo, OR^(e), C(═O)R^(e), C(═O)OR^(e), OC(═O)R^(e), C(═O)N(R^(e))₂,C(═O)NR^(e)SO₂R^(e), N(R^(e))₂, NR^(e)C(═O)R^(e), NR^(e)C(═O)N(R^(e))₂,CN, NO₂, CF₃, OCF₃, SR^(e), SOR^(e), SO₂N(R^(e))₂, and OSO₂CF₃; R⁷ isselected from the group consisting of hydrogen, C₁₋₆alkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl; and n is 1, 2, 3, 4, or 5, or apharmaceutically acceptable salt or hydrate thereof and wherein theintraarterial administration is intraarterial infusion.
 15. The methodof claim 14, wherein the compound is:


16. The method of claim 14, wherein the breast cancer has metastasizedto the liver.
 17. The method of claim 14, wherein the intraarterialadministering occurs at a frequency of one to four times per day. 18.The method of claim 17, wherein the intraarterial administering occursat a frequency of one time per day.
 19. The method of claim 14, whereinthe intraarterial administering occurs at a frequency of one time perweek.